EP1257580A2 - Human ion channels - Google Patents
Human ion channelsInfo
- Publication number
- EP1257580A2 EP1257580A2 EP01910631A EP01910631A EP1257580A2 EP 1257580 A2 EP1257580 A2 EP 1257580A2 EP 01910631 A EP01910631 A EP 01910631A EP 01910631 A EP01910631 A EP 01910631A EP 1257580 A2 EP1257580 A2 EP 1257580A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- ion
- seq
- nucleic acid
- sequence
- polypeptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
Definitions
- the present invention is directed, in part, to nucleic acid molecules encoding ion channels, the novel polypeptides of these human ion channels, and assays for screening compounds that bind to these polypeptides and/or modulate their activities.
- Ion channels are "molecular gates” that regulate the flow of ions into and out of cells. Ion flow plays an important role in all brain cell communication necessary for learning and memory. Additionally, ion flow is important in many physiological processes including, but not limited to, heart rate and body movement. Abe ⁇ ations in ion channels have been implicated in, amongst other disorders, epilepsy, schizophrenia, Alzheimer's disease, migraine, arrhythmia, diabetes, and stroke damage. Ions flow down their electrochemical gradient tlirough the ion channels (passive transport). The core of the channel is hydrophilic, and contains a part of the protein, the selectivity filter, which recognizes only certain ions and allows them to pass through. Channels are named by the ion(s) they allow to pass.
- ion channels include, but are not limited to, calcium channels, potassium channels, sodium channels, chloride channels, etc.
- An additional component of the channel is the gate. Only when the gate is open can the ions recognized by the selectivity filter pass through the channel. Gates open in response to a variety of stimuli, including, but not limited to, changes in membrane potential or the presence of certain chemicals outside or inside the cell. Channel names often also include an indication of what controls the gate: e.g., "voltage-gated calcium channel.” Presently, more than 50 different types of ion channels have been identified.
- Neurotransmitters Communication between neurons is achieved by the release of neurotransmitters into the synapse. These neurotransmitters then activate receptors on the post-synaptic neuron. Many such receptors contain pores to rapidly conduct ions, such as sodium, calcium, potassium, and chloride, into the neuron. These pores, or channels, are made of protein subunits that are members of the family of proteins generally referred to as neurotransmitter- gated ion channel proteins.
- GABA gamma-aminobutyric-acid
- acetylcholine receptor protein subunits including alpha-9 chain, epsilon chain, and beta-2 chain.
- the neurotransmitter-gated ion channel superfamily includes 5-HT3, GABA A , glutamate, glycine, and nicotinic acetylcholine receptor families. Within this superfamily, functional receptors are formed by homo- or heteropentamers of subunits having four transmembrane domains and an extracellular ligand-binding domain. The transmembrane domains of these receptors contribute to the formation of an ion pore.
- Serotonin also known as 5-hydroxytryptamine or 5-HT
- 5-HT is a biogenic amine that functions as a neurotransmitter, a mitogen and a hormone
- Serotonin activates a large number of receptors, most of which are coupled to activation of G-proteins.
- 5-HT3 receptors are structurally distinct and belong to the neurotransmitter-gated ion channel superfamily. 5-HT3 receptors are expressed both pre- and post-synaptically on central and peripheral neurons.
- Post-synaptic 5-HT3 receptors achieve their effects by inducing excitatory potentials in the post-synaptic neuron, whereas pre-synaptic 5-HT3 receptors modulate the release of other neurotransmitters from the pre-synaptic neuron (Conley, 1995).
- 5-HT3 receptors have important roles in pain reception, cognition, cranial motor neuron activity, sensory processing and modulation of affect (Conley, 1995).
- ligands or drugs that modulate 5-HT3 receptors may be useful in treating pain, neuropathies, migraine, cognitive disorders, learning and memory deficits, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, emesis, cranial neuropathies, sensory deficits, anxiety, depression, schizophrenia, and other affective disorders.
- Nicotinic acetylcholine receptors are distinguished from other acetylcholine receptors by their affinity for nicotine and their structure — homo- or hetero-pentamers like all members of the neurotransmitter-gated ion channel superfamily. Nicotinic AChRs are found at the neuromuscular junction on skeletal muscle and on peripheral and central neurons.
- Nicotinic AChRs are receptors for anesthetics, sedatives, and hallucinogens (Conley, 1995), and certain ligands have shown improvements in learning and memory in animals (Levin et al, Behavioral Pharmacology, 1999, 10:675-780).
- ligands or drugs that modulate nicotinic AChRs could be useful for anesthesia, sedation, improving learning and memory, improving cognition, schizophrenia, anxiety, depression, attention deficit hyperactivity disorder, and addiction or smoking cessation.
- Expression of AChR subunits is regulated during development enabling the design of ligands or drugs specifically targeted for particular developmental stages or diseases.
- GABA neurotransmitter ⁇ -aminobutyric acid
- GABA A neurotransmitter-gated ion channels
- GABA B G protein-coupled receptors
- GABAA receptors form chloride channels that induce inhibitory or hyperpolarizing currents when stimulated by GABA or GABA A receptor agonists (Conley, 1995).
- GABA A receptors are modulated by benzodiazepines, barbiturates, picrotoxin, and bicucuilline (Conley, 1995).
- ligands or drugs that modulate GABA A receptors could be useful in sedation, anxiety, epilepsy, seizures, alcohol addiction or withdrawal, panic disorders, pre-menstrual syndrome, migraine, and other diseases characterized by hyper- excitability of central or peripheral neurons.
- the pharmacology of GABA A receptors is affected by changing the subunit composition of the receptor.
- GABA receptor rho subunits are relatively specifically expressed in the retina (Cutting et al, 1991, Proc. Natl. Acad. Sci. USA, 88:2673-7), and the pharmacology of rho receptor homomultimers resembles that of so-called GABA C receptors (Shimada et al, 1992, Mol. Pharmacol. 41:683-7). Therefore, GABA receptors consisting of rho subunits may be useful targets for discovering ligands or drugs to treat visual defects, macular degeneration, glaucoma, and other retinal disorders.
- Compounds that modify the activity of these channels may also be useful for the control of neuromotor diseases including epilepsy and neurodegenerative diseases including Parkinson's and Alzheimer's.
- compounds that modulate the activity of these channels may treat diseases including but not limited to cardiovascular a ⁇ hythmias, stroke, and endocrine and muscular disorders. Therefore, ion channels may be useful targets for discovering ligands or drugs to treat many diverse disorders and defects, including schizophrenia, depression, anxiety, attention deficit hyperactivity disorder, migraine, stroke, ischemia, and neurodegenerative disease such as Alzheimer's disease, Parkinson's disease, glaucoma and maculardegeneration.
- compounds which modulate ion channels can be used for the treatment of cardiovascular diseases including ischemia, congestive heart failure, a ⁇ hythmia, high blood pressure and restenosis.
- the present invention relates to an isolated nucleic acid molecule that comprises a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence homologous to a sequence selected from the group consisting of SEQ ID NO: 8 to SEQ ID NO: 14, or a fragment thereof.
- the nucleic acid molecule encodes at least a portion of ion-x (wherein x is 8, 9, 10, 11, 12, 13, and 14).
- the nucleic acid molecule comprises a sequence that encodes a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO:8 to SEQ ID NO: 14, or a fragment thereof.
- the nucleic acid molecule comprises a sequence homologous to a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:7, or a fragment thereof. In some embodiments, the nucleic acid molecule comprises a sequence selected from the group consisting of SEQ ID NO:l to SEQ ID NO: 7, and fragments thereof.
- the present invention provides vectors which comprise the nucleic acid molecule of the invention.
- the vector is an expression vector.
- the present invention provides host cells which comprise the vectors of the invention.
- the host cells comprise expression vectors.
- the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence complementary to at least a portion of a sequence selected from the group consisting of SEQ ID NO:l to SEQ ID NO:7, said portion comprising at least 10 nucleotides.
- the present invention provides a method of producing a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 8 to SEQ ID NO: 14, or a homolog or fragment thereof.
- the method comprising the steps of introducing a recombinant expression vector that includes a nucleotide sequence that encodes the polypeptide into a compatible host cell, growing the host cell under conditions for expression of the polypeptide and recovering the polypeptide.
- the present invention provides an isolated antibody which binds to an epitope on a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO:8 to SEQ ID NO: 14, or a homolog or fragment thereof.
- the present invention provides an method of inducing an immune response in a mammal against a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO:8 to SEQ ID NO: 14, or a homolog or fragment thereof.
- the method comprises administering to a mammal an amount of the polypeptide sufficient to induce said immune response.
- the present invention provides a method for identifying a compound which binds ion- x.
- the method comprises the steps of: contacting ion-x with a compound and determining whether the compound binds ion-x.
- the present invention provides a method for identifying a compound which binds a nucleic acid molecule encoding ion-x.
- the method comprises the steps of contacting said nucleic acid molecule encoding ion-x with a compound and determining whether said compound binds said nucleic acid molecule.
- the present invention provides a method for identifying a compound which modulates the activity of ion-x.
- the method comprises the steps of contacting ion-x with a compound and determining whether ion-x activity has been modulated.
- the present invention provides a method of identifying an animal homolog of ion-x.
- the method comprises the steps screening a nucleic acid database of the animal with a sequence selected from the group consisting of SEQ ID NO:l to SEQ ID NO:7, or a portion thereof and determining whether a portion of said library or database is homologous to said sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:7, or portion thereof.
- the present invention provides a method of identifying an animal homolog of ion-x.
- the methods comprises the steps screening a nucleic acid library of the animal with a nucleic acid molecule having a sequence selected from the group consisting of SEQ ID NO:l t> SEQ ID NO:7, or a portion thereof; and determining whether a portion of said library or database is homologous to said sequence selected from the group consisting of SEQ ID NO:l to SEQ ID NO: 7, or a portion thereof.
- Another aspect of the present invention relates to methods of screening a human subject to diagnose a disorder affecting the brain or genetic predisposition therefor.
- the methods comprise the steps of assaying nucleic acid of a human subject to determine a presence or an absence of a mutation altering an amino acid sequence, expression, or biological activity of at least one ion channel that is expressed in the brain.
- the ion channels comprise an amino acid sequence selected from the group consisting of: SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 12, and SEQ ID NO: 14, and allelic variants thereof.
- a diagnosis of the disorder or predisposition is made from the presence or absence of the mutation.
- the presence of a mutation altering the amino acid sequence, expression, or biological activity of the ion channel in the nucleic acid correlates with an increased risk of developing the disorder.
- the present invention further relates to methods of screening for an ion-8, ion-9, ion- 12, or ion- 14 mental disorder genotype in a human patient.
- the methods comprise the steps of providing a biological sample comprising nucleic acid from the patient, in which the nucleic acid includes sequences co ⁇ esponding to allelles of ion-8, ion-9, ion-12, or ion-14.
- the presence of one or more mutations in the ion-8, ion-9, ion-12, or ion-14 allelle is detected indicative of a mental disorder genotype.
- the mental disorder includes, but is not limited to, schizophrenia, affective disorders, ADHD/ ADD (i.e, Attention Deficit-Hyperactivity Disorder/Attention Deficit Disorder), and neural disorders such as Alzheimer's disease, Parkinson's disease, migraine, and senile dementia as well as depression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses, and the like.
- ADHD/ ADD i.e, Attention Deficit-Hyperactivity Disorder/Attention Deficit Disorder
- neural disorders such as Alzheimer's disease, Parkinson's disease, migraine, and senile dementia as well as depression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses, and the like.
- kits for screening a human subject to diagnose a mental disorder or a genetic predisposition therefor include an oligonucleotide useful as a probe for identifying polymorphisms in a human ion-8, ion-9, ion-12, or ion-14 gene.
- the oligonucleotide comprises 6-50 nucleotides in a sequence that is identical or complementary to a sequence of a wild type human ion-8, ion-9, ion-12, or ion-14 gene sequence or coding sequence, except for one sequence difference selected from the group consisting of a nucleotide addition, a nucleotide deletion, or nucleotide substitution.
- the kit also includes a media packaged with the oligonucleotide.
- the media contains information for identifying polymorphisms that correlate with a mental disorder or a genetic predisposition therefor, the polymorphisms being identifiable using the oligonucleotide as a probe.
- the present invention further relates to methods of identifying ion channel allelic variants that correlates with mental disorders.
- the methods comprise the steps of providing biological samples that comprise nucleic acid from a human patient diagnosed with a mental disorder, or from the patient's genetic progenitors or progeny, and detecting in the nucleic acid the presence of one or more mutations in an io ⁇ channel that is expressed in the brain.
- the ion channel comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 12, and SEQ ID NO: 14, and allelic variants thereof.
- the nucleic acid includes sequences co ⁇ esponding to the gene or genes encoding ion-x.
- the one or more mutations detected indicate an allelic variant that correlates with a mental disorder.
- the present invention further relates to purified polynucleotides comprising nucleotide sequences encoding alleles of ion-8, ion-9, ion-12, or ion-14 from a human with a mental disorder.
- the polynucleotide hybridizes to the complement of SEQ ID NOS: 1, 2, 5, or 7 under the following hybridization conditions: (a) hybridization for 16 hours at 42° C in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate and (b) washing 2 times for 30 minutes at 60°C in a wash solution comprising O.lx SSC and 1% SDS.
- the polynucleotide that encodes ion-8, ion-9, ion-12, or ion-14 amino acid sequence of the human differs from SEQ ID NOS: 1, 2, 5, or 7 by at least one residue.
- the present invention also provides methods for identifying a modulator of biological activity of ion-x comprising the steps of contacting a cell that expresses ion-x in the presence and in the absence of a putative modulator compound and measuring ion-x biological activity in the cell.
- the decreased or increased ion-x biological activity in the presence versus absence of the putative modulator is indicative of a modulator of biological activity.
- biological activity of an ion channel refers to the native activity of the ion channel. Activities of ion channels include, but are not limited to, the ability to bind or be affected by certain compounds, and the ability to transport ions from one side of the membrane to the other side.
- the present invention further provides methods to identify compounds useful for the treatment of mental disorders.
- the methods comprise the steps of contacting a composition comprising ion-8, ion-9, ion-12, or ion-14 with a compound suspected of binding ion-8, ion- 9, ion-12, or ion-14.
- the binding between ion-8, ion-9, ion-12, or ion-14 and the compound suspected of binding ion-8, ion-9, ion-12, or ion-14 and the compound suspected of binding ion-8, ion-9, ion-12, or ion-14 is detected.
- Compounds identified as binding ion-8, ion-9, ion-12, or ion-14 are candidate compounds useful for the treatment of mental disorders.
- the present invention further provides methods for identifying a compound useful as a modulator of binding between ion-x and a binding partner of ion-x.
- the methods comprise the steps of contacting the binding partner and a composition comprising ion-x in the presence and in the absence of a putative modulator compound and detecting binding between the binding partner and ion-x. Decreased or increased binding between the binding partner and ion-x in the presence of the putative modulator, as compared to binding in the absence of the putative modulator is indicative a modulator compound useful for the treatment of mental disorders.
- the present invention further provides chimeric receptors comprising at least a portion of a sequence selected from the group consisting of SEQ ID NO:l to SEQ ID NO:7, said portion comprising at least 10 nucleotides.
- the present invention provides, inter alia, isolated and purified polynucleotides that encode human ion channels or a portion thereof, vectors containing these polynucleotides, host cells transformed with these vectors, processes of making ion channels and subunits, methods of using the above polynucleotides and vectors, isolated and purified ion channels and subunits, methods of screening compounds which modulate ion channel activity, and compounds that modulate ion channel activity. Definitions
- ion channel refers to an entire channel that allows the movement of ions across a membrane, as well as to subunit polypeptide chains that comprise such a channel.
- the ion channels of the present inventions are ligand-gated, the ion channels are also refe ⁇ ed to as "receptors.”
- receptors Those of skill in the art will recognize that ion channels are made of subunits.
- subunit refers to any component portion of an ion channel, including but not limited to the beta subunit and other associated subunits.
- Synchronucleotide as used herein and understood in the art, refers to polynucleotides produced by purely chemical, as opposed to enzymatic, methods. “Wholly” synthesized DNA sequences are therefore produced entirely by chemical means, and “partially” synthesized DNAs embrace those wherein only portions of the resulting DNA were produced by chemical means.
- region is meant a physically contiguous portion of the primary structure of a biomolecule.
- a region is defined by a contiguous portion of the amino acid sequence of that protein.
- domain is herein defined as referring to a structural part of a biomolecule that contributes to a known or suspected function of the biomolecule. Domains may be coextensive with regions or portions thereof; domains may also incorporate a portion of a biomolecule that is distinct from a particular region, in addition to all or part of that region. Examples of ion channel domains include, but are not limited to, the extracellular (i.e., N- terminal), transmembrane and cytoplasmic (i.e., C-terminal) domains, which are co-extensive with like-named regions of ion channels; and each of the loop segments (both extracellular and intracellular loops) connecting adjacent transmembrane segments.
- the term "activity" refers to a variety of measurable indicia suggesting or revealing binding, either direct or indirect; affecting a response, i.e., having a measurable affect in response to some exposure or stimulus, including, for example, the affinity of a compound for directly binding a polypeptide or polynucleotide of the invention.
- Activity can also be determined by measurement of downstream enzyme activities, and downstream messengers such as K + ions, Ca 2+ ions, Na + ions, Cl " ions, cyclic AMP, and phospholipids after some stimulus or event.
- activity can be determined by measuring ion flux.
- the term "ion flux" includes ion current.
- Activity can also be measured by measuring changes in membrane potential using electrodes or voltage-sensitive dyes, or by measuring neuronal or cellular activity such as action potential duration or frequency, the threshold for stimulating action potentials, long-term potentiation, or long-term inhibition.
- protein is intended to include full length and partial fragments of proteins.
- protein may be used, herein, interchangeably with “polypeptide.”
- protein includes polypeptide, peptide, oligopeptide, or amino acid sequence.
- chimeric receptor is intended to refer to a receptor comprising portions of more than one type of receptor.
- a chimeric receptor may comprise the pore-forming transmembrane domain of an alpha9 nicotinic acetylcholine receptor and the extracellular domain of the alpha 10 nicotinic acetylcholine receptor.
- Chimeric receptors of the present invention are not limited to hybrids of related receptors; chimeric receptors may also include, for example, the pore-forming transmembrane domain of an alpha7 nicotinic acetylcholine receptor and the extracellular domain of the GABA receptor.
- Chimeric receptors may also include portions of known wild- type receptors and portions of artificial receptors.
- antibody is meant to refer to complete, intact antibodies, Fab fragments, and F(ab) 2 fragments thereof.
- Complete, intact antibodies include monoclonal antibodies such as murine monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, and recombinant antibodies identified using phage display.
- binding means the physical or chemical interaction between two proteins, compounds or molecules (including nucleic acids, such as DNA or RNA), or combinations thereof. Binding includes ionic, non-ionic, hydrogen bonds, Van der Waals, hydrophobic interactions, etc.
- the physical interaction, the binding can be either direct or indirect, indirect being through or due to the effects of another protein, compound or molecule. Direct binding refers to interactions that do not take place through or due to the effect of another protein, compound or molecule, but instead are without other substantial chemical intermediates. Binding may be detected in many different manners. As a non- limiting example, the physical binding interaction between an ion channel of the invention and a compound can be detected using a labeled compound.
- functional evidence of binding can be detected using, for example, a cell transfected with and expressing an ion channel of the invention. Binding of the transfected cell to a ligand of the ion channel that was transfected into the cell provides functional evidence of binding. Other methods of detecting binding are well known to those of skill in the art.
- the term "compound” means any identifiable chemical or molecule, including, but not limited to a small molecule, peptide, protein, sugar, nucleotide, or nucleic acid. Such compound can be natural or synthetic.
- the term “complementary” refers to Watson-Crick base-pairing between nucleotide units of a nucleic acid molecule.
- the term "contacting" means bringing together, either directly or indirectly, a compound into physical proximity to a polypeptide or polynucleotide of the invention.
- the polypeptide or polynucleotide can be present in any number of buffers, salts, solutions, etc.
- Contacting includes, for example, placing the compound into a beaker, microtiter plate, cell culture flask, or a microarray, such as a gene chip, or the like, which contains either the ion channel polypeptide or fragment thereof, or nucleic acid molecule encoding an ion channel or fragment thereof.
- homologous nucleotide sequence refers to sequences characterized by a homology, at the nucleotide level or amino acid level, of at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90%, and most preferably at least about 95% to the entirety of SEQ ID NO:l to SEQ ID NO:7, or to at least a portion of SEQ ID NO:l to SEQ ID NO: 1, which portion encodes a functional domain of the encoded polypeptide, or to SEQ ID NO:8 to SEQ ID NO: 14.
- Homologous nucleotide sequences include those sequences coding for isoforms of ion channel proteins. Such isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. Homologous nucleotide sequences include nucleotide sequences encoding for an ion channel protein of a species other than human, including, but not limited to, mammals. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. Although the present invention provides particular sequences, it is understood that the invention is intended to include within its scope other human allelic variants and non-human forms of the ion channels described herein.
- Homologous amino acid sequences include those amino acid sequences which contain conservative amino acid substitutions in SEQ ID NO: 8 to SEQ ID NO: 14, as well as polypeptides having ion channel activity.
- a homologous amino acid sequence does not, however, include the sequence of known polypeptides having ion channel activity. Percent homology can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wl), which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489, which is incorporated herein by reference in its entirety) using the default settings.
- percent homology and its variants are used interchangeably with “percent identity” and “percent similarity.”
- isolated nucleic acid molecule refers to a nucleic acid molecule (DNA or RNA) that has been removed from its native environment.
- isolated nucleic acid molecules include, but are not limited to, recombinant DNA molecules contained in a vector, recombinant DNA molecules maintained in a heterologous host cell, partially or substantially purified nucleic acid molecules, and synthetic DNA or RNA molecules.
- the terms “modulates” or “modifies” means an increase or decrease in the amount, quality, or effect of a particular activity or protein.
- preventing refers to decreasing the probability that an organism contracts or develops an abnormal condition.
- treating refers to having a therapeutic effect and at least partially alleviating or abrogating an abnormal condition in the organism.
- a therapeutic effect refers to the inhibition or activation factors causing or contributing to the abnormal condition.
- a therapeutic effect relieves to some extent one or more of the symptoms of the abnormal condition.
- a therapeutic effect can refer to one or more of the following: (a) an increase in the proliferation, growth, and/or differentiation of cells; (b) inhibition (i.e., slowing or stopping) of cell death; (c) inhibition of degeneration; (d) relieving to some extent one or more of the symptoms associated with the abnormal condition; and (e) enhancing the function of the affected population of cells.
- Compounds demonstrating efficacy against abnormal conditions can be identified as described herein.
- abnormal condition refers to a function in the cells or tissues of an organism that deviates from their normal functions in that organism.
- An abnormal condition can relate to cell proliferation, cell differentiation, cell signaling, or cell survival.
- An abnormal condition may also include obesity, diabetic complications such as retinal degeneration, and irregularities in glucose uptake and metabolism, and fatty acid uptake and metabolism.
- Abnormal cell proliferative conditions include cancers such as fibrotic and mesa ⁇ gial disorders, abnormal angiogenesis and vasculogenesis, wound healing, psoriasis, diabetes mellitus, and inflammation.
- Abnormal differentiation conditions include, but are not limited to, neurodegenerative disorders, slow wound healing rates, and slow tissue grafting healing rates.
- Abnormal cell signaling conditions include, but are not limited to, psychiatric disorders involving excess neurotransmitter activity.
- Abnormal cell survival conditions may also relate to conditions in which programmed cell death (apoptosis) pathways are activated or abrogated.
- a number of protein kinases are associated with the apoptosis pathways. Abe ⁇ ations in the function of any one of the protein kinases could lead to cell immortality or premature cell death.
- the term "administering" relates to a method of incorporating a compound into cells or tissues of an organism.
- the abnormal condition can be prevented or treated when the cells or tissues of the organism exist within the organism or outside of the organism. Cells existing outside the organism can be maintained or grown in cell culture dishes.
- many techniques exist in the art to administer compounds including (but not limited to) oral, parenteral, dermal, injection, and aerosol applications.
- multiple techniques exist in the art to administer the compounds, including (but not limited to) cell microinjection techniques, transformation techniques and ca ⁇ ier techniques.
- the abnormal condition can also be prevented or treated by administering a compound to a group of cells having an aberration in ion channel in an organism.
- the effect of administering a compound on organism function can then be monitored.
- the organism is preferably a mouse, rat, rabbit, guinea pig or goat, more preferably a monkey or ape, and most preferably a human.
- amplification it is meant increased numbers of DNA or RNA in a cell compared with normal cells.
- “Amplification” as it refers to RNA can be the detectable presence of RNA in cells, since in some normal cells there is no basal expression of RNA. In other normal cells, a basal level of expression exists, therefore in these cases amplification is the detection of at least 1 to 2-fold, and preferably more, compared to the basal level.
- oligonucleotide refers to a series of linked nucleotide residues which has a sufficient number of bases to be used in a polymerase chain reaction (PCR). This short sequence is based on (or designed from) a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides comprise portions of a nucleic acid sequence having at least about 10 nucleotides and as many as about 50 nucleotides, preferably about 15 to 30 nucleotides. They are chemically synthesized and may be used as probes.
- probe refers to nucleic acid sequences of variable length, preferably between at least about 10 and as many as about 6,000 nucleotides, depending on use. They are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers. They may be single- or double-stranded and are carefully designed to have specificity in PCR, hybridization membrane-based, or ELISA-like technologies.
- stringent hybridization conditions or “stringent conditions” refers to conditions under which a probe, primer, or oligonucleotide will hybridize to its target sequence, but to a minimal number of other sequences Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences will hybridize with specificity to their proper complements at higher temperatures. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH. The T m is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium.
- stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 to 50 nucleotides) and at least about 60°C for longer probes, primers or oligonucleotides.
- Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
- amino acid sequences are presented in the amino (N) to carboxy (C) direction, from left to right.
- the N-terminal ⁇ -amino group and the C-terminal ⁇ -carboxy groups are not depicted in the sequence.
- the nucleotide sequences are presented by single strands only, in the 5' to 3' direction, from left to right.
- Nucleotides and amino acids are represented in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or amino acids are represented by their three letters code designations.
- the present invention provides purified and isolated polynucleotides (e.g., DNA sequences and RNA transcripts, both sense and complementary antisense strands, both single- and double-stranded, including splice variants thereof) that encode unknown ion channels.
- These genes are described herein and designated herein collectively as ion-x (where x is 8, 9, 10, 11, 12, 13, and 14). That is, these genes and gene products are described herein and designated herein as ion-8, ion-9, ion-10, ion-11, ion-12, ion-13, and ion-14.
- Table 1 below identifies the novel gene sequence ion-x designation, the SEQ ID NO: of the gene sequence, and the SEQ ID NO: of the polypeptide encoded thereby.
- Ion-8 (nucleic acid sequence SEQ ID NO:l, amino acid sequence SEQ ID NO:8), ion- 9 (nucleic acid sequence SEQ ID NO:2, amino acid sequence SEQ ID NO:9), ion-12 (nucleic acid sequence SEQ ID NO:5, amino acid sequence SEQ ID NO:12), and ion-14 (nucleic acid sequence SEQ ID NO:7, amino acid sequence SEQ ID NO: 14) have been detected in brain tissue indicating that these ion-x proteins are neuroreceptors.
- the invention provides purified and isolated polynucleotides (e.g., cDNA, genomic DNA, synthetic DNA, RNA, or combinations thereof, whether single- or double-stranded) that comprise a nucleotide sequence encoding the amino acid sequence of the polypeptides of the invention.
- polynucleotides e.g., cDNA, genomic DNA, synthetic DNA, RNA, or combinations thereof, whether single- or double-stranded
- Such polynucleotides are useful for recombinantly expressing the receptor and also for detecting expression of the receptor in cells (e.g., using Northern hybridization and in situ hybridization assays).
- polynucleotides also are useful in the design of antisense and other molecules for the suppression of the expression of ion-x in a cultured cell, a tissue, or an animal; for therapeutic purposes; or to provide a model for diseases or conditions characterized by abe ⁇ ant ion-x expression.
- polynucleotides of the invention are entire isolated, non-recombinant native chromosomes of host cells.
- a preferred polynucleotide has a sequence selected from the group consisting of SEQ ID NO:l to SEQ ID NO: 7, which correspond to naturally occurring ion-x sequences. It will be appreciated that numerous other polynucleotide sequences exist that also encode ion-x having sequence selected from the group consisting of SEQ ID NO: 8 to SEQ ID NO: 14, due to the well-known degeneracy of the universal genetic code.
- the invention also provides a purified and isolated polynucleotide comprising a nucleotide sequence that encodes a mammalian polypeptide, wherein the polynucleotide hybridizes to a polynucleotide having a sequence selected from the group consisting of SEQ ID NO:l to SEQ ID NO:7, or the non-coding strand complementary thereto, under the following hybridization conditions:
- the present invention relates to molecules which comprise the gene sequences that encode the ion channels; constructs and recombinant host cells incorporating the gene sequences; the novel ion-x polypeptides encoded by the gene sequences; antibodies to the polypeptides and homologs; kits employing the polynucleotides and polypeptides, and methods of making and using all of the foregoing.
- the present invention relates to homologs of the gene sequences and of the polypeptides and methods of making and using the same.
- Genomic DNA of the invention comprises the protein-coding region for a polypeptide of the invention and is also intended to include allelic variants thereof. It is widely understood that, for many genes, genomic DNA is transcribed into RNA transcripts that undergo one or more splicing events wherein intron (i.e., non-coding regions) of the transcripts are removed, or "spliced out.” RNA transcripts that can be spliced by alternative mechanisms, and therefore be subject to removal of different RNA sequences but still encode an ion-x polypeptide, are refe ⁇ ed to in the art as splice variants which are embraced by the invention.
- Splice variants comprehended by the invention therefore are encoded by the same original genomic DNA sequences but arise from distinct mRNA transcripts.
- Allelic variants are modified forms of a wild-type gene sequence, the modification resulting from recombination during chromosomal segregation or exposure to conditions which give rise to genetic mutation.
- Allelic variants like wild type genes, are naturally occu ⁇ ing sequences (as opposed to non-naturally occurring variants that arise from in vitro manipulation).
- the invention also comprehends cDNA that is obtained tlirough reverse transcription of an RNA polynucleotide encoding ion-x (conventionally followed by second strand synthesis of a complementary strand to provide a double-stranded DNA).
- a prefe ⁇ ed DNA of the invention comprises a double stranded molecule along with the complementary molecule (the "non-coding strand” or “complement") having a sequence unambiguously deducible from the coding strand according to Watson-Crick base-pairing rules for DNA.
- polynucleotides encoding the ion-x polypeptide of sequences selected from the group consisting of SEQ ID NO:8 to SEQ ID NO: 14, which differ in sequence from the polynucleotides of sequences selected from the group consisting of SEQ ID NO:l to SEQ ID NO:7, by virtue of the well-known degeneracy of the universal nuclear genetic code.
- the invention further embraces other species, preferably mammalian, homologs of the human ion-x DNA.
- Species homologs sometimes refe ⁇ ed to as "orthologs," in general, share at least 35%, at least 40%>, at least 45%, at least 50%, at least 60%>, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% homology with human DNA of the invention.
- percent sequence "homology" with respect to polynucleotides of the invention may be calculated as the percentage of nucleotide bases in the candidate sequence that are identical to nucleotides in the ion-x sequence selected from the group consisting of SEQ ID NO:l to SEQ ID NO:7, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
- Polynucleotides of the invention permit identification and isolation of polynucleotides encoding related ion-x polypeptides, such as human allelic variants and species homologs, by well-known techniques including Southern and/or Northern hybridization, and polymerase chain reaction (PCR).
- related polynucleotides include human and non-human genomic sequences, including allelic variants, as well as polynucleotides encoding polypeptides homologous to ion-x and structurally related polypeptides sharing one or more biological, immunological, and/or physical properties of ion-x.
- Non-human species genes encoding proteins homologous to ion-x can also be identified by Southern and/or PCR analysis and are useful in animal models for ion-x disorders. Knowledge of the sequence of a human ion-x DNA also makes possible through use of Southern hybridization or polymerase chain reaction (PCR) the identification of genomic DNA sequences encoding ion-x expression control regulatory sequences such as promoters, operators, enhancers, repressors, and the like. Polynucleotides of the invention are also useful in hybridization assays to detect the capacity of cells to express ion-x.
- Polynucleotides of the invention may also provide a basis for diagnostic methods useful for identifying a genetic alteration(s) in an ion-x locus that underlies a disease state or states, which information is useful both for diagnosis and for selection of therapeutic strategies.
- the ion-x nucleotide sequences disclosed herein may be used to identify homologs of the ion-x, in other animals, including but not limited to humans and other mammals, and invertebrates.
- nucleotide sequences disclosed herein, or any portion thereof can be used, for example, as probes to screen databases or nucleic acid libraries, such as, for example, genomic or cDNA libraries, to identify homologs, using screening procedures well known to those skilled in the art. Accordingly, homologs having at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 100% homology with ion-x sequences can be identified.
- polynucleotides encoding ion-x polypeptides makes readily available to the worker of ordinary skill in the art many possible fragments of the ion channel polynucleotide.
- Polynucleotide sequences provided herein may encode, as non-limiting examples, a native channel, a constitutive active channel, or a dominant-negative channel.
- One prefe ⁇ ed embodiment of the present invention provides an isolated nucleic acid molecule comprising a sequence homologous to a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:7, and fragments thereof.
- Another prefe ⁇ ed embodiment provides an isolated nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NO:l to SEQ ID NO:7, and fragments thereof.
- fragments of-ion-x-encoding polynucleotides comprise at least 10, and preferably at least 12, 14, 16, 18, 20, 25, 50, or 75 consecutive nucleotides of a polynucleotide encoding ion-x.
- fragment polynucleotides of the invention comprise sequences unique to the ion-x-encoding polynucleotide sequence, and therefore hybridize under highly stringent or moderately stringent conditions only (i.e., "specifically") to polynucleotides encoding ion-x (or fragments thereof).
- Polynucleotide fragments of genomic sequences of the invention comprise not only sequences unique to the coding region, but also include fragments of the full-length sequence derived from introns, regulatory regions, and or other non-translated sequences. Sequences unique to polynucleotides of the invention are recognizable through sequence comparison to other known polynucleotides, and can be identified through use of alignment programs routinely utilized in the art, e.g., those made available in public sequence databases. Such sequences also are recognizable from Southern hybridization analyses to determine the number of fragments of genomic DNA to which a polynucleotide will hybridize.
- Polynucleotides of the invention can be labeled in a manner that permits their detection, including radioactive, fluorescent, and enzymatic labeling. Fragment polynucleotides are particularly useful as probes for detection of full-length or fragments of ion-x polynucleotides.
- One or more polynucleotides can be included in kits that are used to detect the presence of a polynucleotide encoding ion-x, or used to detect variations in a polynucleotide sequence encoding ion-x.
- the invention also embraces DNAs encoding ion-x polypeptides that hybridize under moderately stringent or high stringency conditions to the non-coding strand, orcomplement, of the polynucleotides set forth in a sequence selected from the group consisting of SEQ ID NO:l to SEQ ID NO:7.
- Exemplary highly stringent hybridization conditions are as follows: hybridization at 42°C in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10%> Dextran sulfate, and washing twice for 30 minutes at 60°C in a wash solution comprising 0.1 X SSC and 1% SDS. It is understood in the art that conditions of equivalent stringency can be achieved through variation of temperature and buffer, or salt concentration as described Ausubel et al. (Eds.), Protocols in Molecular Biology, John Wiley & Sons (1994), pp. 6.0.3 to 6.4.10. Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and the percentage of guanosine/cytosine (GC) base pairing of the probe. The hybridization conditions can be calculated as described in Sambrook, et al, (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47 to 9.51.
- nucleotide sequence information disclosed in the present invention, one skilled in the art can identify and obtain nucleotide sequences which encode ion-x from different sources (i.e., different tissues or different organisms) through a variety of means well known to the skilled artisan and as disclosed by, for example, Sambrooket al, "Molecular cloning: a laboratory manual", Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), which is incorporated herein by reference in its entirety.
- DNA that encodes ion-x may be obtained by screening mRNA, cDNA, or genomic DNA with oligonucleotide probes generated from the ion-x gene sequence information provided herein. Probes may be labeled with a detectable group, such as a fluorescent group, a radioactive atom or a chemiluminescent group in accordance with procedures known to the skilled artisan and used in conventional hybridization assays, as described by, for example, Sambrook et al.
- a detectable group such as a fluorescent group, a radioactive atom or a chemiluminescent group
- a nucleic acid molecule comprising any of the ion-x nucleotide sequences described above can alternatively be synthesized by use of the polymerase chain reaction (PCR) procedure, with the PCR oligonucleotide primers produced from the nucleotide sequences provided herein.
- PCR polymerase chain reaction
- the PCR reaction provides a method for selectively increasing the concentration of a particular nucleic acid sequence even when that sequence has not been previously purified and is present only in a single copy in a particular sample.
- the method can be used to amplify either single- or double-stranded DNA.
- the essence of the method involves the use of two oligonucleotide probes to serve as primers for the template-dependent, polymerase mediated replication of a desired nucleic acid molecule.
- Automated sequencing methods can be used to obtain or verify the nucleotide sequence of ion-x.
- the ion-x nucleotide sequences of the present invention are believed to be 100% accurate.
- nucleotide sequence obtained by automated methods may contain some e ⁇ ors.
- Nucleotide sequences determined by automation are typically at least about 90%, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of a given nucleic acid molecule. The actual sequence may be more precisely determined using manual sequencing methods, which are well known in the art.
- An e ⁇ or in a sequence which results in an insertion or deletion of one or more nucleotides may result in a frame shift in translation such that the predicted amino acid sequence will differ from that which would be predicted from the actual nucleotide sequence of the nucleic acid molecule, starting at the point of the mutation.
- nucleic acid molecules of the present invention are useful for screening for restriction fragment length polymorphism (RFLP) associated with certain disorders, as well as for genetic mapping.
- RFLP restriction fragment length polymorphism
- the polynucleotide sequence information provided by the invention makes possible large-scale expression of the encoded polypeptide by techniques well known and routinely practiced in the art.
- vectors or recombinant expression vectors, comprising any of the nucleic acid molecules described above.
- Vectors are used herein either to amplify DNA or RNA encoding ion-x and/or to express DNA which encodes ion-x.
- Prefe ⁇ ed vectors include, but are not limited to, plasmids, phages, cosmids, episomes, viral particles or viruses, and integratable DNA fragments (i.e., fragments integratable into the host genome by homologous recombination).
- Prefe ⁇ ed viral particles include, but are not limited to, adenoviruses, baculoviruses, parvoviruses, herpesviruses, poxviruses, adeno-associated viruses, Semliki Forest viruses, vaccinia viruses, and retroviruses.
- Prefe ⁇ ed expression vectors include, but are not limited to, pcDNA3 (Invitrogen) and pSVL (Pharmacia Biotech).
- expression vectors include, but are not limited to, pSPORTTM vectors, pGEMTM vectors (Promega), pPROEXvectorsTM (LTI, Bethesda, MD), BluescriptTM vectors (Stratagene), pQETM vectors (Qiagen), pSE420TM (Invitrogen), and pYES2TM(Invitrogen).
- Expression constructs preferably comprise ion-x-encoding polynucleotides operatively linked to an endogenous or exogenous expression control DNA sequence and a transcription terminator.
- Expression control DNA sequences include promoters, enhancers, operators, and regulatory element binding sites generally, and are typically selected based on the expression systems in which the expression construct is to be utilized. Prefe ⁇ ed promoter and enhancer sequences are generally selected for the ability to increase gene expression, while operator sequences are generally selected for the ability to regulate gene expression.
- Expression constructs of the invention may also include sequences encoding one or more selectable markers that permit identification of host cells bearing the construct. Expression constructs may also include sequences that facilitate, and preferably promote, homologous recombination in a host cell. Prefe ⁇ ed constructs of the invention also include sequences necessary for replication in a host cell.
- Expression constructs are preferably utilized for production of an encoded protein, but may also be utilized simply to amplify an ion-x-encoding polynucleotide sequence.
- the vector is an expression vector wherein the polynucleotide of the invention is operatively linked to a polynucleotide comprising an expression control sequence.
- Autonomously replicating recombinant expression constructs such as plasmid and viral DNA vectors incorporating polynucleotides of the invention are also provided.
- Prefe ⁇ ed expression vectors are replicable DNA constructs in which a DNA sequence encoding ion-x is operably linked or connected to suitable control sequences capable of effecting the expression of the ion-x in a suitable host.
- DNA regions are operably linked or connected when they are functionally related to each other.
- a promoter is operably linked or connected to a coding sequence if it controls the transcription of the sequence.
- Amplification vectors do not require expression control domains, but rather need only the ability to replicate in a host, usually confe ⁇ ed by an origin of replication, and a selection gene to facilitate recognition of transformants.
- the need for control sequences in the expression vector will vary depending upon the host selected and the transformation method chosen.
- control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal • binding and sequences which control the termination of transcription and translation.
- Prefe ⁇ ed vectors preferably contain a promoter that is recognized by the host organism.
- the promoter sequences of the present invention may be prokaryotic, eukaryotic or viral.
- suitable prokaryotic sequences include the P R and P promoters of bacteriophage lambda (The bacteriophage Lambda, Hershey, A. D., Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1973), which is incorporated herein by reference in its entirety; Lambda II, Hendrix, R. W., Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1980), which is incorporated herein by reference in its entirety); the tip, recA, heat shock, and lacZ promoters of E.
- Additional promoters include, but are not limited to, mouse mammary tumor virus, long terminal repeat of human immunodeficiency virus, maloney virus, cytomegalovirus immediate early promoter, Epstein Ban virus, Rous sarcoma virus, human actin, human myosin, human hemoglobin, human muscle creatine, and human metalothionein.
- Additional regulatory sequences can also be included in prefe ⁇ ed vectors.
- Prefe ⁇ ed examples of suitable regulatory sequences are represented by the Shine-Dalgarno of the replicase gene of the phage MS-2 and of the gene ell of bacteriophage lambda.
- the Shine- Dalgarno sequence may be directly followed by DNA encoding ion-x and result in the expression of the mature ion-x protein.
- suitable expression vectors can include an appropriate marker that allows the screening of the transformed host cells.
- the transformation of the selected host is carried out using any one of the various techniques well known to the expert in the art and described in Sambrook et al, supra.
- An origin of replication can also be provided either by construction of the vector to include an exogenous origin or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter may be sufficient.
- a selectable marker is dihydrofolate reductase (DHFR) or thymidine kinase (see, U.S. Patent No. 4,399,216).
- Nucleotide sequences encoding ion-x may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulation are disclosed by Sambrook et al, supra and are well known in the art. Methods for construction of mammalian expression vectors are disclosed in, for example, Okayamaet al, Mol Cell Biol, 1983,3, 280, Cosman et al, Mol. Immunol, 1986, 23, 935, Cosman et al, Nature, 1984, 312, 768, EP-A-0367566, and WO 91/18982, each of which is incorporated herein by reference in its entirety. Host cells
- host cells including prokaryotic and eukaryotic cells, comprising a polynucleotide of the invention (or vector of the invention) in a manner that permits expression of the encoded ion-x polypeptide.
- Polynucleotides of the invention may be introduced into the host cell as part of a circular plasmid, or as linear DNA comprising an isolated protein coding region or a viral vector.
- Methods for introducing DNA into the host cell that are well known and routinely practiced in the art include transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts.
- Expression systems of the invention include bacterial, yeast, fungal, plant, insect, invertebrate, vertebrate, and mammalian cells systems.
- the invention provides host cells that are transformed or transfected (stably or transiently) with polynucleotides of the invention or vectors of the invention. As stated above, such host cells are useful for amplifying the polynucleotides and also for expressing the ion-x polypeptide or fragment thereof encoded by the polynucleotide.
- the invention provides a method for producing an ion-x polypeptide (or fragment thereof) comprising the steps of growing a host cell of the invention in a nutrient medium and isolating the polypeptide or variant thereof from the cell or the medium.
- ion-x is a membrane spanning channel
- the preferable isolation may involve isolation of cell membranes containing the polypeptide embedded therein, whereas for other applications a more complete isolation may be preferable.
- transformed host cells having an expression vector comprising any of the nucleic acid molecules described above are provided. Expression of the nucleotide sequence occurs when the expression vector is introduced into an appropriate host cell.
- Suitable host cells for expression of the polypeptides of the invention include, but are not limited to, prokaryotes, yeast, and eukaryotes. If a prokaryotic expression vector is employed, then the appropriate host cell would be any prokaryotic cell capable of expressing the cloned sequences. Suitable prokaryotic cells include, but are not limited to, bacteria of the genera Escherichia, Bacillus, Salmonella, Pseudomonas, Streptomyces, and Staphylococcus.
- eukaryotic cells are cells of higher eukaryotes.
- Suitable eukaryotic cells include, but are not limited to, non-human mammalian tissue culture cells and human tissue culture cells.
- Prefe ⁇ ed host cells include, but are not limited to, insect cells, HeLa cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (COS cells), human 293 cells, and murine 3T3 fibroblasts. Propagation of such cells in cell culture has become a routine procedure (see, Tissue Culture, Academic Press, Kruse and Patterson, eds. (1973), which is incorporated herein by reference in its entirety).
- a yeast host may be employed as a host cell.
- Prefe ⁇ ed yeast cells include, but are not limited to, the genera Saccharomyces, Pichia, and Kluveromyces.
- Prefe ⁇ ed yeast hosts are S. cerevisiae and P. pastoris.
- Prefe ⁇ ed yeast vectors can contain an origin of replication sequence from a 2T yeast plasmid, an autonomously replication sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene.
- ARS autonomously replication sequence
- Shuttle vectors for replication in both yeast and E. coli are also included herein.
- insect cells may be used as host cells.
- the polypeptides of the invention are expressed using a baculovirus expression system (see, Luckow et al, Bio/Technology, 1988, 6, 47, Baculovirus Expression Vectors: A Laboratory Manual, O'Rielly et al. (Eds.), W.H. Freeman and Company, New York, 1992, and U.S. Patent No. 4,879,236, each of which is incorporated herein by reference in its entirety).
- the MAXBACTM complete baculovirus expression system can, for example, be used for production in insect cells.
- Host cells of the invention are a valuable source of immunogen for development of antibodies specifically immunoreactive with ion-x.
- Host cells of the invention are also useful in methods for the large-scale production of ion-x polypeptides wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells, or from the medium in which the cells are grown, by purification methods known in the art, e.g., conventional chromatographic methods including immunoaffinity chromatography, receptor affinity chromatography, hydrophobic interaction chromatography, lectin affinity chromatography, size exclusion filtration, cation or anion exchange chromatography, high pressure liquid chromatography (HPLC), reverse phase HPLC, and the like.
- HPLC high pressure liquid chromatography
- Still other methods of purification include those methods wherein the desired protein is expressed and purified as a fusion protein having a specific tag, label, or chelating moiety that is recognized by a specific binding partner or agent.
- the purified protein can be cleaved to yield the desired protein, or can be left as an intact fusion protein. Cleavage of the fusion component may produce a form of the desired protein having additional amino acid residues as a result of the cleavage process.
- ion-x DNA sequences allows for modification of cells to permit, or increase, expression of endogenous ion-x.
- Cells can be modified (e.g., by homologous recombination) to provide increased expression by replacing, in whole or in part, the naturally occurring ion-x promoter with all or part of a heterologous promoter so that the cells express ion-x at higher levels.
- the heterologous promoter is inserted in such a manner that it is operatively linked to endogenous ion-x encoding sequences.
- amplifiable marker DNA e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamoyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase
- intron DNA may be inserted along with the heterologous promoter DNA. If linked to the ion-x coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the ion-x coding sequences in the cells. Knock-outs
- the DNA sequence information provided by the present invention also makes possible the development (e.g., by homologous recombination or "knock-out” strategies; see Capecchi, Science 244: 1288-1292 (1989), which is incorporated herein by reference) of animals that fail to express functional ion-x or that express a variant of ion-x.
- animals especially small laboratory animals such as rats, rabbits, and mice
- anti-sense polynucleotides that recognize and hybridize to polynucleotides encoding ion-x.
- Full-length and fragment anti-sense polynucleotides are provided.
- Fragment antisense molecules of the invention include (i) those that specifically recognize and hybridize to ion-x RNA (as determined by sequence comparison of DNA encoding ion-x to DNA encoding other known molecules). Identification of sequences unique to ion-x encoding polynucleotides can be deduced through use of any publicly available sequence database, and/or tlirough use of commercially available sequence comparison programs.
- Anti-sense polynucleotides are particularly relevant to regulating expression of ion-x by those cells expressing ion-x mRNA.
- Antisense nucleic acids preferably 10 to 30 base-pair oligonucleotides capable of specifically binding to ion-x expression control sequences or ion-x RNA are introduced into cells (e.g., by a viral vector or colloidal dispersion system such as a liposome).
- the antisense nucleic acid binds to the ion-x target nucleotide sequence in the cell and prevents transcription and/or translation of the target sequence.
- Phosphorothioate and methylphosphonate antisense oligonucleotides are specifically contemplated for therapeutic use by the invention.
- Locked nucleic acids are also specifically contemplated for therapeutic use by the present invention.
- the antisense oligonucleotides may be further modified by adding poly-L-lysine, transferrin polylysine, or cholesterol moieties at their 5' end. Suppression of ion-x expression at either the transcriptional or translational level is useful to generate cellular or animal models for diseases/conditions characterized by abe ⁇ ant ion-x expression.
- Antisense oligonucleotides, or fragments of nucleotide sequences selected from the group consisting of SEQ ID NO:l to SEQ ID NO: 7, or sequences complementary or homologous thereto, derived from the nucleotide sequences of the present invention encoding ion-x are useful as diagnostic tools for probing gene expression in various tissues.
- tissue can be probed in situ with oligonucleotide probes carrying detectable groups by conventional autoradiography techniques to investigate native expression of this enzyme or pathological conditions relating thereto.
- Antisense oligonucleotides are preferably directed to regulatory regions of sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:7, or mRNA co ⁇ esponding thereto, including, but not limited to, the initiation codon, TATA box, enhancer sequences, and the like. Transcription factors
- the ion-x sequences taught in the present invention facilitate the design of novel transcription factors for modulating ion-x expression in native cells and animals, and cells transformed or transfected with ion-x polynucleotides.
- the Cys 2 -His 2 zinc finger proteins which bind DNA via their zinc finger domains, have been shown to be amenable to structural changes that lead to the recognition of different target sequences.
- These artificial zinc finger proteins recognize specific target sites with high affinity and low dissociation constants, and are able to act as gene switches to modulate gene expression.
- Knowledge of the particular ion-x target sequence of the present invention facilitates the engineering of zinc finger proteins specific for the target sequence using knownmethods such as a combination of structure-based modeling and screening of phage display libraries (Segal et al, Proc. Natl. Acad. Sci. (USA) 96:2758-2763 (1999); Liu et al, Proc. Natl. Acad. Sci. (USA) 94:5525-5530 (1997); Greisman et /., Science 275:657-661 (1997); Choo et al, J. Mol. Biol. 273:525-532 (1997)).
- Each zinc finger domain usually recognizes three or more base pairs.
- a zinc finger protein consisting of 6 tandem repeats of zinc fingers would be expected to ensure specificity for a particular sequence (Segal et al)
- the artificial zinc finger repeats designed based on ion-x sequences, are fused to activation or repression domains to promote or suppress ion-x expression (Liuet al.)
- the zinc finger domains can be fused to the TATA box-binding factor (TBP) with varying lengths of linker region between the zinc finger peptide and the TBP to create either transcriptional activators or repressors (Kim et al, Proc. Natl. Acad. Sci.
- Such proteins and polynucleotides that encode them have utility for modulating ion-x expression in vivo in both native cells, animals and humans; and/or cells transfected with ion-x- encoding sequences.
- the novel transcription factor can be delivered to the target cells by transfecting constructs that express the transcription factor (gene therapy), or by introducing the protein.
- Engineered zinc finger proteins can also be designed to bind RNA sequences for use in therapeutics as alternatives to antisense or catalytic RNA methods (McCollet al, Proc. Natl. Acad. Sci. (USA) 96:9521-9526 (1997); Wuet al, Proc. Natl. Acad. Sci.
- the present invention contemplates methods of designing such transcription factors based on the gene sequence of the invention, as well as customized zinc finger proteins, that are useful to modulate ion-x expression in cells (native or transformed) whose genetic complement includes these sequences.
- the invention also provides purified and isolated mammalian ion-x polypeptides encoded by a polynucleotide of the invention.
- a human ion-x polypeptide comprising the amino acid sequence set out in sequences selected from the group consisting of SEQ ID NO:8 to SEQ ID NO: 14, or fragments thereof comprising an epitope specific to the polypeptide.
- epitope specific to is meant a portion of the ion-x receptor that is recognizable by an antibody that is specific for the ion-x, as defined in detail below.
- sequences provided are particular human sequences, the invention is intended to include within its scope other human allelic variants; non-human mammalian fonns of ion-x, and other vertebrate forms of ion-x.
- the invention provides a purified and isolated polypeptide comprising at least one extracellular domain of ion-x.
- Purified and isolated polypeptides comprising the extracellular domain of ion-x are highly prefe ⁇ ed.
- Such fragments may be continuous portions of the native receptor.
- knowledge of the ion-x gene and protein sequences as provided herein permits recombining of various domains that are not contiguous in the native protein.
- ion-x was shown to contain transmembrane-spanning domains.
- the invention also embraces polypeptides that have at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55% or at least 50%> identity and/or homology to the prefe ⁇ ed polypeptide of the invention.
- Percent amino acid sequence "identity" with respect to the prefe ⁇ ed polypeptide of the invention is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the ion-x sequence after aligning both sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
- Percent sequence "homology" with respect to the prefe ⁇ ed polypeptide of the invention is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the ion-x sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and also considering any conservative substitutions as part of the sequence identity.
- percent homology is calculated as the percentage of amino acid residues in the smaller of two sequences which align with identical amino acid residue in the sequence being compared, when four gaps in a length of 100 amino asids may be introduced to maximize alignment [Dayhoff, in Atlas of Protein Sequence and Structure, Vol. 5, p. 124, National Biochemical Research Foundation, Washington, D.C. (1972), incorporated herein by reference].
- Polypeptides of the invention may be isolated from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving host cells of the invention. Use of mammalian host cells is expected to provide for such post-translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention. Glycosylated and non-glycosylated forms of ion-x polypeptides are embraced by the invention.
- the invention also embraces variant (or analog) ion-x polypeptides.
- insertion variants are provided wherein one or more amino acid residues supplement an ion-x amino acid sequence. Insertions may be located at either or both termini of the protein, or may be positioned within internal regions of the ion-x amino acid sequence. Insertional variants with additional residues at either or both termini can include, for example, fusion proteins and proteins including amino acid tags or labels.
- Insertion variants include ion-x polypeptides wherein one or more amino acid residues are added to an ion-x acid sequence or to a biologically active fragment thereof.
- Variant products of the invention also include mature ion-x products, i.e., ion-x products wherein leader or signal sequences are removed, with additional amino terminal residues.
- the additional amino terminal residues may be derived from another protein, or may include one or more residues that are not identifiable as being derived from specific proteins.
- Ion-x products with an additional methionine residue at position-1 are contemplated, as are variants with additional methionine and lysine residues at positions -2 and -1 (Mef 2 -Lys _1 -ion-x).
- Variants of ion-x with additional Met, Met-Lys, Lys residues are particularly useful for enhanced recombinant protein production in bacterial host cells.
- the invention also embraces ion-x variants having additional amino acid residues that result from use of specific expression systems.
- use of commercially available vectors that express a desired polypeptide as part of a glutathione-S-transferase (GST) fusion product provides the desired polypeptide having an additional glycine residue at position -1 after cleavage of the GST component from the desired polypeptide.
- GST glutathione-S-transferase
- Insertional variants also include fusion proteins wherein the amino terminus and/or the carboxy terminus of ion-x is/are fused to another polypeptide.
- the invention provides deletion variants wherein one or more amino acid residues in an ion-x polypeptide are removed.
- Deletions can be effected at one or both termini of the ion-x polypeptide, or with removal of one or more non-terminal amino acid residues of ion-x.
- Deletion variants therefore, include all fragments of an ion-x polypeptide.
- the invention also embraces polypeptide fragments of sequences selected from the group consisting of SEQ ID NO: 8 to SEQ ID NO: 14, wherein the fragments maintain biological (e.g., ligand binding and/or ion trafficking) and/or immunological properties of a ion-x polypeptide.
- an isolated nucleic acid molecule comprises a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence homologous to a sequence selected from the group consisting of SEQ ID NO: 8 to SEQ ID NO: 14, and fragments thereof, wherein the nucleic acid molecule encodes at least a portion of ion-x.
- the isolated nucleic acid molecule comprises a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:7, and fragments thereof.
- polypeptide fragments comprise at least 5, 10, 15, 20, 25, 30, 35, or 40 consecutive amino acids of a sequence selected from the group consisting of SEQ ID NO:8 to SEQ ID NO: 14.
- Prefe ⁇ ed polypeptide fragments display antigenic properties unique to, or specific for, human ion-x and its allelic and species homologs. Fragments of the invention having the desired biological and immunological properties can be prepared by any of the methods well known and routinely practiced in the art.
- the nucleic acid molecule comprises SEQ ID NO: 1.
- the nucleic acid molecule comprises a fragment of SEQ ID NO: 1.
- the invention provides fragments of SEQ ID NO:l which comprise at least 14 and preferably at least 16, 18, 20, 25, 50, or 75 consecutive nucleotides.
- the fragment can be located within any portion of SEQ ID NO: 1, may include more than one portion of SEQ ID NO: 1 , or may include repeated portions of SEQ ID NO: 1.
- the nucleic acid molecule comprises a sequence related to alpha7 nicotinic AChR.
- the nucleic acid molecule comprises SEQ ID NO:2.
- the nucleic acid molecule comprises a fragment of SEQ ID NO:2.
- the invention provides fragments of SEQ ID NO:2 which comprise at least 14 and preferably at least 16, 18, 20, 25, 50, or 75 consecutive nucleotides.
- the fragment can be located within any portion of SEQ ID NO:2, may include more than one portion of SEQ ID NO:2, or may include repeated portions of SEQ ID NO:2.
- the nucleic acid molecule comprises a sequence related to the acetylcholine receptor, non-alpha-3 chain precursor.
- the nucleic acid molecule comprises SEQ ID NO:3.
- the nucleic acid molecule comprises a fragment of SEQ ID NO:3.
- the invention provides fragments of SEQ ID NO: 3 which comprise at least 14 and preferably at least 16, 18, 20, 25, 50, or 75 consecutive nucleotides.
- the fragment can be located within any portion of SEQ ID NO: 3, may include more than one portion of SEQ ID NO: 3, or may include repeated portions of SEQ ID NO:3.
- the nucleic acid molecule comprises a sequence related to the acetylcholine receptor, beta-3 chain precursor.
- the nucleic acid molecule comprises SEQ ID NO:4.
- the nucleic acid molecule comprises a fragment of SEQ ID NO:4.
- the invention provides fragments of SEQ ID NO:4 which comprise at least 14 and preferably at least 16, 18, 20, 25, 50, or 75 consecutive nucleotides.
- the fragment can be located within any portion of SEQ ID NO:4, may include more than one portion of SEQ ID NO:4, or may include repeated portions of SEQ ID NO:4.
- the nucleic acid molecule comprises a sequence related to GABA rho-3 subunit precursor.
- the nucleic acid molecule comprises SEQ ID NO:5.
- the nucleic acid molecule comprises a fragment of SEQ ID NO:5.
- the invention provides fragments of SEQ ID NO: 5 which comprise at least 14 and preferably at least 16, 18, 20, 25, 50, or 75 consecutive nucleotides.
- the fragment can be located within any portion of SEQ ID NO: 5, may include more than one portion of SEQ ID NO: 5, or may include repeated portions of SEQ ID NO: 5.
- the nucleic acid molecule comprises a sequence related to the acetylcholine receptor, alpha-9 chain precursor.
- the nucleic acid molecule comprises SEQ ID NO:6.
- the nucleic acid molecule comprises a fragment of SEQ ID NO: 6.
- the invention provides fragments of SEQ ID NO: 6 which comprise at least 14 and preferably at least 16, 18, 20, 25, 50, or 75 consecutive nucleotides.
- the fragment can be located within any portion of SEQ ID NO: 6, may include more than one portion of SEQ ID NO:6, or may include repeated portions of SEQ ID NO:6.
- the nucleic acid molecule comprises a sequence related to the glutamate receptor, ionotropic kainate 4 precursor.
- the nucleic acid molecule comprises SEQ ID NO:7.
- the nucleic acid molecule comprises a fragment of SEQ ID NO: 7.
- the invention provides fragments of SEQ ID NO: 7 which comprise at least 14 and preferably at least 16, 18, 20, 25, 50, or 75 consecutive nucleotides.
- the fra g ment can be located within any portion of SEQ ID NO: 7, may include more than one portion of SEQ ID NO:7, or may include repeated portions of SEQ ID NO:7.
- the nucleic acid molecule comprises a sequence related to the acetylcholine receptor, alpha chain precursor.
- the invention provides substitution variants of ion-x polypeptides.
- Substitution variants include those polypeptides wherein one or more amino acid residues of an ion-x polypeptide are removed and replaced with alternative residues.
- the substitutions are conservative in nature; however, the invention embraces substitutions that are also non-conservative. Conservative substitutions for this purpose may be defined as set out in Tables 2, 3, or 4 below.
- Variant polypeptides include those wherein conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of the invention.
- Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure.
- a conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties.
- Exemplary conservative substitutions are set out in Table 2 (from WO 97/09433, page 10, published March 13, 1997 (PCT/GB96/02197, filed 9/6/96), immediately below. Table 2
- conservative amino acids can be grouped as described in Lehninger, [Biochemistry, Second Edition; Worth Publishers, Inc. NY, NY (1975), pp.71-77] as set out in Table 3, below.
- polypeptides of the invention is intended to include polypeptides bearing modifications other than insertion, deletion, or substitution of amino acid residues.
- the modifications may be covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic, and inorganic moieties.
- Such derivatives may be prepared to increase circulating half-life of a polypeptide, or may be designed to improve the targeting capacity of the polypeptide for desired cells, tissues, or organs.
- the invention further embraces ion-x polypeptides that have been covalently modified to include one or more water-soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.
- Variants that display ligand binding properties of native ion-x and are expressed at higher levels are particularly useful in assays of the invention; the variants are also useful in providing cellular, tissue and animal models of diseases/conditions characterized by abe ⁇ ant ion-x activity.
- compositions comprising purified polypeptides of the invention.
- Prefe ⁇ ed compositions comprise, in addition to the polypeptide of the invention, a pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semisolid, or solid diluent that serves as a pharmaceutical vehicle, excipient, or medium. Any diluent known in the art may be used.
- Exemplary diluents include, but are not limited to, water, saline solutions, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- and propylhydroxybenzoate, talc, alginates, starches, lactose, sucrose, dextrose, sorbitol, mannitol, glycerol, calcium phosphate, mineral oil, and cocoa butter.
- Variants that display ligand binding properties of native ion-x and are expressed at higher levels are particularly useful in assays of the invention; the variants are also useful in assays of the invention and in providing cellular, tissue and animal models of diseases/conditions characterized by abe ⁇ ant ion-x activity.
- antibodies e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds which include CDR sequences which specifically recognize a polypeptide of the invention) specific for ion-x or fragments thereof.
- CDR complementary determining region
- Prefe ⁇ ed antibodies of the invention are human antibodies that are produced and identified according to methods described in W093/11236, published June 20, 1993, which is incorporated herein by reference in its entirety.
- Antibody fragments, including Fab, Fab', F(ab') 2 , and F v are also provided by the invention.
- variable regions of the antibodies of the invention recognize and bind ion-x polypeptides exclusively (i.e., are able to distinguish ion-x polypeptides from other known ion channel polypeptides by virtue of measurable differences in binding affinity, despite the possible existence of localized sequence identity, homology, or similarity between ion-x and such polypeptides).
- specific antibodies may also interact with other proteins (for example, S. aureusp ⁇ otein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and, in particular, in the constant region of the molecule.
- the invention provides an antibody that is specific for the ion-x of the invention.
- Antibody specificity is described in greater detail below.
- antibodies that can be generated from polypeptides that have previously been described in the literature and that are capable of fortuitously cross-reacting with ion-x are considered “cross-reactive" antibodies.
- Such cross-reactive antibodies are not antibodies that are "specific" for ion-x.
- the determination of whether an antibody is specific for ion-x or is cross-reactive with another known receptor is made using any of several assays, such as Western blotting assays, that are well known in the art.
- antibodies that specifically bind to an extracellular epitope of the ion-x are prefe ⁇ ed.
- the invention provides monoclonal antibodies. Hybridomas that produce such antibodies also are intended as aspects of the invention.
- the invention provides a humanized antibody. Humanized antibodies are useful for in vivo therapeutic indications.
- the invention provides a cell-free composition comprising polyclonal antibodies, wherein at least one of the antibodies is an antibody of the invention specific for ion-x.
- Antisera isolated from an animal is an exemplary composition, as is a composition comprising an antibody fraction of an antisera that has been resuspended in water or in another diluent, excipient, or ca ⁇ ier.
- the invention provides an anti-idiotypic antibody specific for an antibody that is specific for ion-x.
- antibodies contain relatively small antigen binding domains that can be isolated chemically or by recombinant techniques. Such domains are useful ion-x binding molecules themselves, and also may be reintroduced into human antibodies, or fused to toxins or other polypeptides.
- the invention provides a polypeptide comprising a fragment of an ion-x-specific antibody, wherein the fragment and the polypeptide bind to the ion-x.
- the invention provides polypeptides that are single chain antibodies and CDR-grafted antibodies.
- Non-human antibodies may be humanized by any of the methods known in the art.
- the non-human CDRs are inserted into a human antibody or consensus antibody framework sequence. Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity.
- Antibodies of the invention are useful for, e.g., therapeutic purposes (by modulating activity of ion-x), diagnostic purposes to detect or quantitate ion-x, and purification of ion-x.
- Kits comprising an antibody of the invention for any of the purposes described herein are also comprehended.
- a kit of the invention also includes a control antigen for which the antibody is immunospecific.
- the invention comprehends gene therapy to restore ion-x activity to treat those disease states.
- Delivery of a functional ion-x gene to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), ⁇ ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments). See, for example, Anderson, Nature, supplement to vol. 392, No. 6679, pp.25-20 (1998).
- compositions including pharmaceutical compositions, comprising any of the nucleic acid molecules or recombinant expression vectors described above and an acceptable ca ⁇ ier or diluent.
- the carrier or diluent is pharmaceutically acceptable.
- Suitable ca ⁇ iers are described in the most recent edition of Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field, which is incorporated herein by reference in its entirety.
- Prefe ⁇ ed examples of such ca ⁇ iers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils may also be used. The formulations are sterilized by commonly used techniques.
- compositions comprising polypeptides, polynucleotides, or antibodies of the invention that have been formulated with, e.g., a pharmaceutically acceptable carrier.
- the invention also provides methods of using antibodies of the invention.
- the invention provides a method for modulating ligand binding of an ion-x comprising the step of contacting the ion-x with an antibody specific for the ion-x, under conditions wherein the antibody binds the receptor.
- Ion channels that may be expressed in the brain provide an indication that abe ⁇ ant ion-x signaling activity may co ⁇ elate with one or more neurological or psychological disorders.
- the invention also provides a method for treating a neurological or psychiatric disorder comprising the step of administering to a mammal in need of such treatment an amount of an antibody-like polypeptide of the invention that is sufficient to modulate ligand binding to an ion-x in neurons of the mammal.
- Ion-x may also be expressed in many tissues, including but not limited to, kidney, colon, small intestine, stomach, testis, placenta, adrenal gland, peripheral blood leukocytes, bone ma ⁇ ow, retina, ovary, fetal brain, fetal liver, heart, spleen, liver, lung, muscle, thyroid gland, uterus, prostate, skin, salivary gland, and pancreas. Tissues where specific ion-x of the present invention are expressed are identified in Example 11 , below. Kits
- kits including pharmaceutical kits.
- the kits can comprise any of the nucleic acid molecules described above, any of the polypeptides described above, or any antibody which binds to a polypeptide of the invention as described above, as well as a negative control.
- the kit preferably comprises additional components, such as, for example, instructions, solid support, reagents helpful for quantification, and the like.
- the invention features methods for detection of a polypeptide in a sample as a diagnostic tool for diseases or disorders, wherein the method comprises the steps of: (a) contacting the sample with a nucleic acid probe which hybridizes under hybridization assay conditions to a nucleic acid target region of a polypeptide having a sequence selected from the group consisting of SEQ ID NO:8 to SEQ ID NO: 14, said probe comprising the nucleic acid sequence encoding the polypeptide, fragments thereof, and the complements of the sequences and fragments; and (b) detecting the presence or amount of the probe:target region hybrid as an indication of the disease.
- the disease is selected from the group consisting of thyroid disorders (e.g. thyreotoxicosis, myxoedema); renal failure; inflammatory conditions (e.g., Crohn's disease); diseases related to cell differentiation and homeostasis; rheumatoid arthritis; autoimmune disorders; movement disorders; CNS disorders (e.g., pain including neuropathic pain, migraine, and other headaches; stroke; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, anxiety, generalized anxiety disorder, post-traumatic-stress disorder, depression, bipolar disorder, delirium, dementia, severe mental retardation; dyskinesias, such as Huntington's disease or Tourette's Syndrome; attention disorders including ADD and ADHD, and degenerative disorders such as Parkinson's, Alzheimer's; movement disorders, including ataxias, supranuclear palsy, etc.); infections, such as viral infections caused by HIV1 or HlV-2; metabolic and cardiovascular diseases and disorders (e.g., type), HIV-1 or HlV
- kits may be designed to detect either expression of polynucleotides encoding these proteins or the proteins themselves in order to identify tissue as being neurological.
- oligonucleotide hybridization kits can be provided which include a container having an oligonucleotide probe specific for the ion-x-specific DNA and optionally, containers with positive and negative controls and/or instructions.
- PCR kits can be provided which include a container having primers specific for the ion-x-specific sequences, DNA and optionally, containers with size markers, positive and negative controls and/or instructions.
- Hybridization conditions should be such that hybridization occurs only with the genes in the presence of other nucleic acid molecules. Under stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having 1 or 2 mismatches out of 20 contiguous nucleotides. Such conditions are defined supra.
- the diseases for which detection of genes in a sample could be diagnostic include diseases in which nucleic acid (DNA and/or RNA) is amplified in comparison to normal cells.
- amplification is meant increased numbers of DNA or RNA in a cell compared with normal cells.
- the diseases that could be diagnosed by detection of nucleic acid in a sample preferably include central nervous system and metabolic diseases.
- the test samples suitable for nucleic acid probing methods of the present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids.
- the samples used in theabove-described methods will vary based on the assay format, the detection method and the nature of the tissues, cells or extracts to be assayed. Methods for preparing nucleic acid extracts of cells are well known in the art and can be readily adapted in order to obtain a sample that is compatible with the method utilized.
- immunoassay kits can be provided which have containers container having antibodies specific for the ion-x protein and optionally, containers with positive and negative controls and/or instructions.
- Kits may also be provided useful in the identification of ion-x binding partners such as natural ligands, neurotransmitters, or modulators (agonists or antagonists).
- Substances useful for treatment of disorders or diseases preferably show positive results in one or more in vitro assays for an activity co ⁇ esponding to treatment of the disease or disorder in question.
- Substances that modulate the activity of the polypeptides preferably include, but are not limited to, antisense oligonucleotides, agonists and antagonists, and inhibitors of protein kinases.
- Another aspect of the present invention is directed to methods of inducing an immune response in a mammal against a polypeptide of the invention by administering to the mammal an amount of the polypeptide sufficient to induce an immune response.
- the amount will be dependent on the animal species, size of the animal, and the like but can be determined by those skilled in the art.
- the invention also provides assays to identify compounds that bind ion-x.
- One such assay comprises the steps of: (a) contacting a composition comprising an ion-x with a compound suspected of binding ion-x; and (b) measuring binding between the compound and ion-x.
- the composition comprises a cell expressing ion-x on its surface.
- isolated ion-x or cell membranes comprising ion-x are employed.
- the binding may be measured directly, e.g., by using a labeled compound, or may be measured indirectly by several techniques, including measuring ion trafficking of ion-x induced by the compound.
- binding molecules including natural ligands and synthetic compounds, can be identified or developed using isolated or recombinant ion-x products, ion-x variants, or preferably, cells expressing such products. Binding partners are useful for purifying ion-x products and detection or quantification of ion-x products in fluid and tissue samples using known immunological procedures. Binding molecules are also manifestly useful in modulating (i.e., blocking, inhibiting or stimulating) biological activities of ion-x, especially those activities involved in signal transduction.
- the DNA and amino acid sequence information provided by the present invention also makes possible identification of binding partner compounds with which an ion-x polypeptide or polynucleotide will interact.
- Methods to identify binding partner compounds include solution assays, in vitro assays wherein ion-x polypeptides are immobilized, and cell based assays. Identification of binding partner compounds of ion-x polypeptides provides candidates for therapeutic or prophylactic intervention in pathologies associated with ion-x normal and abe ⁇ ant biological activity.
- the invention includes several assay systems for identifying ion-x-binding partners.
- methods of the invention comprise the steps of (a) contacting an ion-x polypeptide with one or more candidate binding partner compounds and (b) identifying the compounds that bind to the ion-x polypeptide. Identification of the compounds that bind the ion-x polypeptide can be achieved by isolating the ion-x polypeptide/binding partner complex, and separating the binding partner compound from the ion-x polypeptide. An additional step of characterizing the physical, biological, and/or biochemical properties of the binding partner compound is also comprehended in another embodiment of the invention.
- the ion-x polypeptide/binding partner complex is isolated using an antibody immunospecific for either the ion-x polypeptide or the candidate binding partner compound.
- either the ion-x polypeptide or the candidate binding partner compound comprises a label or tag that facilitates its isolation
- methods of the invention to identify binding partner compounds include a step of isolating the ion-x polypeptide/binding partner complex through interaction with the label or tag.
- An exemplary tag of this type is a poly-histidine sequence, generally around six histidine residues, that permits isolation of a compound so labeled using nickel chelation.
- Other labels and tags such as the FLAG ® tag (Eastman Kodak, Rochester, NY), well known and routinely used in the art, are embraced by the invention.
- the invention provides a method comprising the steps of (a) contacting an immobilized ion-x polypeptide with a candidate binding partner compound and (b) detecting binding of the candidate compound to the ion-x polypeptide.
- the candidate binding partner compound is immobilized and binding of ion-x is detected. Immobilization is accomplished using any of the methods well known in the art, including covalent bonding to a support, a bead, or a chromatographic resin, as well as non-covalent, high affinity interactions such as antibody binding, or use of streptavidin/biotin binding wherein the immobilized compound includes a biotin moiety.
- Detection of binding can be accomplished (i) using a radioactive label on the compound that is not immobilized, (ii) using of a fluorescent label on the non-immobilized compound, (iii) using an antibody immunospecific for the non-immobilized compound, (iv) using a label on the non-immobilized compound that excites a fluorescent support to which the immobilized compound is attached, as well as other techniques well known and routinely practiced in the art.
- the invention also provides cell-based assays to identify binding partner compounds of an ion-x polypeptide.
- the invention provides a method comprising the steps of contacting an ion-x polypeptide expressed on the surface of a cell with a candidate binding partner compound and detecting binding of the candidate binding partner compound to the ion-x polypeptide.
- the detection comprises detecting a calcium flux or other physiological event in the cell caused by the binding of the molecule.
- Another aspect of the present invention is directed to methods of identifying compounds that bind to either ion-x or nucleic acid molecules encoding ion-x, comprising contacting ion-x, or a nucleic acid molecule encoding the same, with a compound, and determining whether the compound binds ion-x or a nucleic acid molecule encoding the same.
- Binding can be determined by binding assays which are well known to the skilled artisan, including, but not limited to, gel-shift assays, Western blots, radiolabeled competition assay, phage-based expression cloning, co-fractionation by chromatography, co-precipitation, cross linking, interaction trap/two-hybrid analysis, southwestern analysis, ELISA, and the like, which are described in, for example, Current Protocols in Molecular Biology, 1999, John Wiley & Sons, NY, which is incorporated herein by reference in its entirety.
- the compounds to be screened include (which may include compounds which are suspected to bind ion-x, or a nucleic acid molecule encoding the same), but are not limited to, extracellular, intracellular, biologic or chemical origin.
- the methods of the invention also embrace ligands, especially neuropeptides, that are attached to a label, such as a radiolabel
- ion-x polypeptide or polynucleotide employed in such a test may either be free in solution, attached to a solid support, borne on a cell surface or located intracellularly or associated with a portion of a cell.
- One skilled in the art can, for example, measure the formation of complexes between ion-x and the compound being tested. Alternatively, one skilled in the art can examine the diminution in complex formation between ion-x and its substrate caused by the compound being tested.
- high throughput screening for compounds having suitable binding affinity to ion-x is employed. Briefly, large numbers of different small peptide test compounds are synthesized on a solid substrate. The peptide test compounds are contacted with ion-x and washed. Bound ion-x is then detected by methods well known in the art. Purified polypeptides of the invention can also be coated directly onto plates for use in the aforementioned drug screening techniques. In addition, nonneutralizing antibodies can be used to capture the protein and immobilize it on the solid support.
- an expressed ion-x can be used for HTS binding assays in conjunction with its defined ligand, in this case the co ⁇ esponding neuropeptide that activates it.
- the identified peptide is labeled with a suitable radioisotope, including, but not limited to, 1 5 1, 3 H, 35 S or 32 P, by methods that are well known to those skilled in the art.
- the peptides may be labeled by well-known methods with a suitable fluorescent derivative (Baindur et al, Drug Dev. Res., 1994, 33, 373-398; Rogers, Drug Discovery Today, 1997, 2, 156-160).
- Radioactive ligand specifically bound to the receptor in membrane preparations made from the cell line expressing the recombinant protein can be detected in HTS assays in one of several standard ways, including filtration of the receptor-ligand complex to separate bound ligand from unbound ligand (Williams, Med. Res. Rev., 1991, 11, 147-184; Sweetnam et al, J. Natural Products, 1993, 56, 441-455).
- Alternative methods include a scintillation proximity assay (SPA) or a FlashPlate format in which such separation is unnecessary (Nakayama, Cur. Opinion Drug Disc. Dev., 1998,7, 85-91 Bosse et /., J. Biomolecular Screening, 1998, 3, 285-292.).
- Binding of fluorescent ligands can be detected in various ways, including fluorescence energy transfer (FRET), direct spectrophotofluorometric analysis of bound ligand, or fluorescence polarization (Rogers, Drug Discovery Today, 1997, 2, 156-160; Hill, Cur. Opinion OrugDisc. Dev., 1998, 1, 92-97).
- FRET fluorescence energy transfer
- Differophotofluorometric analysis of bound ligand or fluorescence polarization
- assays may be used to identify specific ligands of a ion-x receptor, including assays that identify ligands of the target protein through measuring direct binding of test ligands to the target protein, as well as assays that identify ligands of target proteins through affinity ultrafiltration with ion spray mass spectroscopy/HPLC methods or other physical and analytical methods.
- binding interactions are evaluated indirectly using the yeast two-hybrid system described in Fields et al, Nature, 340:245-246 (1989), and Fields et al, Trends in Genetics, 10:286-292 (1994), both of which are incorporated herein by reference.
- the two-hybrid system is a genetic assay for detecting interactions between two proteins or polypeptides.
- the two-hybrid system exploits the ability of a pair of interacting proteins to bring a transcription activation domain into close proximity with a DNA binding domain that binds to an upstream activation sequence (UAS) of a reporter gene, and is generally performed in yeast.
- UAS upstream activation sequence
- the assay requires the construction of two hybrid genes encoding (1) a DNA-binding domain that is fused to a first protein and (2) an activation domain fused to a second protein.
- the DNA-binding domain targets the first hybrid protein to the UAS of the reporter gene; however, because most proteins lack an activation domain, this DNA-binding hybrid protein does not activate transcription of the reporter gene.
- the second hybrid protein which contains the activation domain, cannot by itself activate expression of the reporter gene because it does not bind the UAS.
- both hybrid proteins are present, the noncovalent interaction of the first and second proteins tethers the activation domain to the UAS, activating transcription of the reporter gene.
- the first protein is an ion channel gene product, or fragment thereof, that is known to interact with another protein or nucleic acid
- this assay can be used to detect agents that interfere with the binding interaction. Expression of the reporter gene is monitored as different test agents are added to the system. The presence of an inhibitory agent results in lack of a reporter signal.
- the yeast two-hybrid assay can also be used to identify proteins that bind to the gene product.
- a fusion polynucleotide encoding both an ion-x receptor (or fragment) and a UAS binding domain i.e., a first protein
- a large number of hybrid genes each encoding a different second protein fused to an activation domain are produced and screened in the assay.
- the second protein is encoded by one or more members of a total cDNA or genomic DNA fusion library, with each second protein-coding region being fused to the activation domain.
- This system is applicable to a wide variety of proteins, and it is not even necessary to know the identity or function of the second binding protein.
- the system is highly sensitive and can detect interactions not revealed by other methods; even transient interactions may trigger transcription to produce a stable mRNA that can be repeatedly translated to yield the reporter protein.
- test ligands may be used to search for agents that bind to the target protein.
- One such screening method to identify direct binding of test ligands to a target protein is described in U.S. Patent No. 5,585,277, incorporated herein by reference. This method relies on the principle that proteins generally exist as a mixture of folded and unfolded states, and continually alternate between the two states.
- the target protein molecule bound by the ligand remains in its folded state.
- the folded target protein is present to a greater extent in the presence of a test ligand which binds tie target protein, than in the absence of a ligand. Binding of the ligand to the target protein can be determined by any method that distinguishes between the folded and unfolded states of the target protein. The function of the target protein need not be known in order for this assay to be performed. Virtually any agent can be assessed by this method as a test ligand, including, but not limited to, metals, polypeptides, proteins, lipids, polysaccharides, polynucleotides and small organic molecules.
- inventions comprise using competitive screening assays in which neutralizing antibodies capable of binding a polypeptide of the invention specifically compete with a test compound for binding to the polypeptide.
- tie antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with ion-x.
- Radiolabeled competitive binding studies are described in A.H. Lin et al Antimicrobial Agents and Chemotherapy, 1997, vol. 41, no. 10. pp. 2127-2131, the disclosure of which is incorporated herein by reference in its entirety.
- ion-8 nucleic acid sequence SEQ ID NO:l, amino acid sequence SEQ ID NO: 8
- ion-9 nucleic acid sequence SEQ ID NO:2, amino acid sequence SEQ ID NO:9
- ion-12 nucleic acid sequence SEQ ID NO: 5, amino acid sequence SEQ ID NO: 12
- ion-14 nucleic acid sequence SEQ ID NO:7, amino acid sequence SEQ ID NO: 14
- the invention also provides methods for identifying a modulator of binding between a ion-x and an ion-x binding partner, comprising the steps of: (a) contacting an ion-x binding partner and a composition comprising an ion-x in the presence and in the absence of a putative modulator compound; (b) detecting binding between the binding partner and the ion- x; and (c) identifying a putative modulator compound or a modulator compound in view of decreased or increased binding between the binding partner and the ion-x in the presence of the putative modulator, as compared to binding in the absence of the putative modulator.
- Ion-x binding partners that stimulate ion-x activity are useful as agonists in disease states or conditions characterized by insufficient ion-x signaling (e.g., as a result of insufficient activity of an ion-x ligand).
- Ion-x binding partners that block ligand-mediated ion-x signaling are useful as ion-x antagonists to treat disease states or conditions characterized by excessive ion-x signaling.
- ion-x modulators in general, as well as ion-x polynucleotides and polypeptides are useful in diagnostic assays for such diseases or conditions.
- the invention provides methods for treating a disease or abnormal condition by administering to a patient in need of such treatment a substance that modulates the activity or expression of a polypeptide having a sequence selected from the group consisting of SEQ ID NO:8 to SEQ ID NO: 14.
- Agents that modulate (i.e., increase, decrease, or block) ion-x activity or expression may be identified by incubating a putative modulator with a cell containing an ion-x polypeptide or polynucleotide and determining the effect of the putative modulator on ion-x activity or expression.
- the selectivity of a compound that modulates the activity of ion-x can be evaluated by comparing its effects on ion-x to its effect on other ion channel compounds.
- Selective modulators may include, for example, antibodies and other proteins, peptides, or organic molecules that specifically bind to an ion-x polypeptide or an ion-x-encoding nucleic acid.
- Ion-x polynucleotides, polypeptides, and modulators may be used in the treatment of such diseases and conditions as infections, such as viral infections caused by HIV-1 or HIV-2; thyroid disorders (e.g.
- thyreotoxicosis myxoedema
- renal failure inflammatory conditions (e.g., Crohn's disease); diseases related to cell differentiation and homeostasis; rheumatoid arthritis; autoimmune disorders; movement disorders; CNS disorders (e.g., pain including neuropathic pain, migraine, and other headaches; stroke; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, anxiety, generalized anxiety disorder, post-traumatic-stress disorder, depression, bipolar disorder, delirium, dementia, severe mental retardation; dyskinesias, such as Huntington's disease or Tourette's Syndrome; attention disorders including ADD and ADHD, and degenerative disorders such as Parkinson's, Alzheimer's; movement disorders, including ataxias, supranuclear palsy, etc); infections, such as viral infections caused by HIV-l or HIV-2; metabolic and cardiovascular diseases and disorders (e.g., type 2 diabetes, obesity, anorexia, hypotension, hypertension, thrombosis, myocardial in
- Methods of the invention to identify modulators include variations on any of the methods described above to identify binding partner compounds, the variations including techniques wherein a binding partner compound has been identified and the binding assay is ca ⁇ ied out in the presence and absence of a candidate modulator.
- a modulator is identified in those instances where binding between the ion-x polypeptide and tfie binding partner compound changes in the presence of the candidate modulator compared to binding in the absence of the candidate modulator compound.
- a modulator that increases binding between the ion-x polypeptide and the binding partner compound is described as an enhancer or activator, and a modulator that decreases binding between the ion-x polypeptide and the binding partner compound is described as an inhibitor.
- the invention also comprehends high-throughput screening (HTS) assays to identify compounds that interact with or inhibit biological activity (i.e., affect enzymatic activity, binding activity, etc.) of an ion-x polypeptide.
- HTS assays permit screening of large numbers of compounds in an efficient manner.
- Cell-based HTS systems are contemplated to investigate ion-x receptor-ligand interaction.
- HTS assays are designed to identify "hits” or "lead compounds” having the desired property, from which modifications can be designed to improve the desired property. Chemical modification of the "hit” or "lead compound” is often based on an identifiable structure/activity relationship between the "hit” and the ion-x polypeptide.
- Another aspect of the present invention is directed to methods of identifying compounds which modulate (i.e., increase or decrease) activity of ion-x comprising contacting ion-x with a compound, and determining whether the compound modifies activity of ion-x.
- the activity in the presence of the test compared is measured to the activity in the absence of the test compound.
- One of skill in the art can, for example, measure the activity of the ion channel polypeptide using electrophysiological methods, described infra. Where the activity of the sample containing the test compound is higher than the activity in the sample lacking the test compound, the compound will have increased activity. Similarly, where the activity of the sample containing the test compound is lower than the activity in the sample lacking the test compound, the compound will have inhibited activity.
- the activity of the polypeptides of the invention can also be determined by, as non- limiting examples, the ability to bind or be activated by certain ligands, including, but not limited to, known neurotransmitters, agonists and antagonists, including but not limied to serotonin, acetylcholine, nicotine, and GABA.
- the activity of the ion channels can be assayed by examining activity such as ability to bind or be affected by calcium ions, hormones, chemokines, neuropeptides, neurotransmitters, nucleotides, lipids, odorants, and photons.
- the assay may take the form of an ion flux assay, a membrane potential assay, a yeast growth assay, a cAMP assay, an inositol triphosphate assay, a diacylglycerol assay, an Aequorin assay, a Luciferase assay, a FLIPR assay for intracellular Ca 2+ concentration, a mitogenesis assay, a MAP Kinase activity assay, an arachidonic acid release assay (e.g., using [ 3 H]-arachidonic acid), and an assay for extracellular acidification rates, as well as other binding or function-based assays of activity that are generally known in the art
- electrophysiology Another potentially useful assay to examine the activity of ion channels is electrophysiology, the measurement of ion permeability across the cell membrane. This technique is described in, for example, Electrophysiology, A Practical Approach, DI Wallis editor, IRL Press at Oxford University Press, (1993), and Voltage and patch Clamping with Microelectrodes, Smith et al, eds., Waverly Press, Inc for the Amercan Physiology Society (1985), each of which is incorporated by reference in its entirety.
- Another assay to examine the activity of ion channels is tlirough the use of the FLIPR Fluorometric Imaging Plate Reader system, developed by Dr. Vince Groppi of the Pharmacia Corporation to perform cell-based, high-throughput screening (HTS) assays measuring, for example, membrane potential. Changes in plasma membrane potential co ⁇ elate with the modulation of ion channels as ions move into or out of the cell.
- the E IPR system measures such changes in membrane potential. This is accomplished by loading cells expressing an ion channel gene with a cell-membrane permeant fluorescent indicator dye suitable for measuring changes in membrane potential such as diBAC (bis-(l,3-dibutylbarbituric acid) pentamethine oxonol, Molecular Probes).
- diBAC bis-(l,3-dibutylbarbituric acid
- the modulation of ion channel activity can be assessed with FLIPR and detected as changes in the emission spectrum of the diBAC dye.
- the present invention is particularly useful for screening compounds by using ion-x in any of a variety of drug screening techniques.
- the compounds to be screened include (which may include compounds which are suspected to modulate ion-x activity), but are not limited to, extracellular, intracellular, biologic or chemical origin.
- the ion-x polypeptide employed in such a test may be in any form, preferably, free in solution, attached to a solid support, borne on a cell surface or located intracellularly.
- One skilled in the art can, for example, measure the formation of complexes between ion-x and the compound being tested. Alternatively, one skilled in the art can examine the diminution in complex formation between ion-x and its substrate caused by the compound being tested.
- the activity of ion-x polypeptides of the invention can be determined by, for example, examining the ability to bind or be activated by chemically synthesized peptide ligands.
- the activity of ion-x polypeptides can be assayed by examining their ability to bind calcium ions, hormones, chemokines, neuropeptides, neurotransmitters, nucleotides, lipids, odorants, and photons.
- the activity of the ion-x polypeptides can be determined by examining the activity of effector molecules including, but not limited to, adenylate cyclase, phospholipases and ion channels.
- modulators of ion-x polypeptide activity may alter ion channel function, such as a binding property of a channel or an activity such as ion selectivity.
- the assay may take the fonn of an ion flux assay, a yeast growth assay, a cAMP assay, an inositol triphosphate assay, a diacylglycerol assay, an Aequorin assay, a Luciferase assay, a FLIPR assay for intracellular Ca 2+ concentration, a mitogenesis assay, a MAP Kinase activity assay, an arachidonic acid release assay (e.g., using [ H]-arachidonic acid), and an assay for extracellular acidification rates, as well as other binding or function-based assays of ion-x activity that are generally known in the art.
- Ion-x activity can be determined by methodologies that are used to assay for FaRP activity, which is well known to those skilled in the art.
- Biological activities of ion-x receptors according to the invention include, but are not limited to, the binding of a natural or an unnatural ligand, as well as any one of the functional activities of ion channels known in the art.
- the modulators of the invention exhibit a variety of chemical structures, which can be generally grouped into non-peptide mimetics of natural ion channel ligands, peptide and non- peptide allosteric effectors of ion channels, and peptides that may function as activators or inhibitors (competitive, uncompetitive and non-competitive) (e.g., antibody products) of ion channels.
- the invention does not restrict the sources for suitable modulators, which may be obtained from natural sources such as plant, animal or mineral extracts, or non-natural sources such as small molecule libraries, including the products of combinatorial chemical approaches to library construction, and peptide libraries.
- organic modulators of ion channels are GABA, serotonin, acetylcholine, nicotine, glutamate, glycine, NMDA, and kainic acid.
- enzyme Assays can be used to examine enzymatic activity including, but not limited to, photometric, radiometric, HPLC, electrochemical, and the like, which are described in, for example, Enzyme Assays : A Practical Approach, eds., R. Eisenthal and M. J. Danson, 1992, Oxford University Press, which is incorporated herein by reference in its entirety.
- Recombinant receptors are prefe ⁇ ed for binding assay HTS because they allow for better specificity (higher relative purity), provide the ability to generate large amounts of receptor material, and can be used in a broad variety of formats (see Hodgson, Bio/Technology, 1992, 10, 973-980; each of which is incorporated herein by reference in its entirety).
- heterologous systems are available for functional expression of recombinant receptors that are well known to those skilled in the art.
- Such systems include bacteria (Strosberg, et al, Trends in Pharmacological Sciences, 1992, 13, 95-98), yeast (Pausch, Trends in Biotechnology, 1997, 15, 487-494), several kinds of insect cells (Vanden Broeck, Int. Rev. Cytology, 1996, 164, 189-268), amphibian cells (Jayawickreme et al, Current Opinion in Biotechnology, 1997, 8, 629-634) and several mammalian cell lines (CHO, HEK293, COS, etc.; see Gerhardt, et al, ⁇ ur. J. Pharmacology, 1997, 334, 1-23).
- These examples do not preclude the use of other possible cell expression systems, including cell lines obtained from nematodes (PCT application WO 98/37177).
- methods of screening for compounds that modulate ion-x activity comprise contacting test compounds with ion-x and assaying for the presence of a complex between the compound and ion-x.
- the ligand is typically labeled. After suitable incubation, free ligand is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular compound to bind to ion-x.
- Such biological responses include, but are not limited to, the following: the ability to survive in the absence of a limiting nutrient in specifically engineered yeast cells (Pausch, Trends in Biotechnology, 1997, 15, 487-494); changes in intracellular Ca 2+ concentration as measured by fluorescent dyes (Murphy, et al, Cur. Opinion Drug Disc. Dev., 1998, 1, 192-199). Fluorescence changes can also be used to monitor ligand-induced changes in membrane potential or intracellular pH; an automated system suitable for HTS has been described for these purposes (Schroeder, et al, J. Biomolecular Screening, 1996, 1, 75- 80).
- permanently transfected CHO cells could be used for the preparation of membranes which contain significant amounts of the recombinant receptor proteins; these membrane preparations would then be used in receptor binding assays, employing the radiolabeled ligand specific for the particular receptor.
- a functional assay such as fluorescent monitoring of ligand-induced changes in internal Ca 2+ concentration or membrane potential in permanently transfected CHO cells containing each of these receptors individually or in combination would be prefe ⁇ ed for HTS.
- Equally prefe ⁇ ed would be an alternative type of mammalian cell, such as HEK293 or COS cells, in similar formats. More prefe ⁇ ed would be permanently transfected insect cell lines, such as Drosophila S2 cells.
- the invention contemplates a multitude of assays to screen and identify inhibitors of ligand binding to ion-x.
- the ion-x is immobilized and interaction with a binding partner is assessed in the presence and absence of a candidate modulator such as an inhibitor compound.
- interaction between the ion-x and its binding partner is assessed in a solution assay, both in the presence and absence of a candidate inhibitor compound.
- an inhibitor is identified as a compound that decreases binding between the ion-x and its binding partner.
- Another contemplated assay involves a variation of the dihybrid assay wherein an inhibitor of protein/protein interactions is identified by detection of a positive signal in a transformed or transfected host cell, as described in PCT publication number WO 95/20652, published August 3, 1995.
- Candidate modulators contemplated by the invention include compounds selected from libraries of either potential activators or potential inhibitors. There are a number of different libraries used for the identification of small molecule modulators, including: (1) chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules. Chemical libraries consist of random chemical structures, some of which are analogs of known compounds or analogs of compounds that have been identified as "hits" or "leads" in other drug discovery screens, some of which are derived from natural products, and some of which arise from non-directed synthetic organic chemistry.
- Natural product libraries are collections of microorganisms, animals, plants, or marine organisms which are used to create mixtures for screening by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of plants or marine organisms. Natural product libraries include polyketides, non- ribosomal peptides, and variants (non-naturally occurring) thereof. For a review, see Science 282:63-68 (1998). Combinatorial libraries are composed of large numbers of peptides, oligonucleotides, or organic compounds as a mixture. These libraries are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning, or proprietary synthetic methods. Of particular interest are non-peptide combinatorial libraries.
- Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries.
- combinatorial chemistry and libraries created therefrom see Myers, Cu ⁇ . Opin. Biotechnol. 8:701-707 (1997). Identification of modulators through use of the various libraries described herein permits modification of the candidate "hit” (or “lead") to optimize the capacity of the "hit" to modulate activity.
- binding partners can be designed and include soluble forms of binding partners, as well as such binding partners as chimeric, or fusion, proteins.
- polypeptides of the invention are employed as a research tool for identification, characterization and purification of interacting, regulatory proteins.
- Appropriate labels are incorporated into the polypeptides of the invention by various methods known in the art and the polypeptides are used to capture interacting molecules. For example, molecules are incubated with the labeled polypeptides, washed to remove unbound polypeptides, and the polypeptide complex is quantified. Data obtained using different concentrations of polypeptide are used to calculate values for the number, affinity, and association of polypeptide with the protein complex.
- Labeled polypeptides are also useful as reagents for the purification of molecules with which the polypeptide interacts including, but not limited to, inhibitors.
- affinity purification a polypeptide is covalently coupled to a chromatography column. Cells and their membranes are extracted, and various cellular subcomponents are passed over the column. Molecules bind to the column by virtue of their affinity to the polypeptide. The polypeptide-complex is recovered from the column, dissociated and the recovered molecule is subjected to protein sequencing. This amino acid sequence is then used to identify the captured molecule or to design degenerate oligonucleotides for cloning the co ⁇ esponding gene from an appropriate cDNA library.
- compounds may be identified which exhibit similar properties to the ligand for the ion-x of the invention, but which are smaller and exhibit a longer half time than the endogenous ligand in a human or animal body.
- a molecule according to the invention is used as a "lead” compound.
- the design of mimetics to known pharmaceutically active compounds is a well-known approach in the development of pharmaceuticals based on such "lead” compounds. Mimetic design, synthesis and testing are generally used to avoid randomly screening a large number of molecules for a target property.
- structural data deriving from the analysis of the deduced amino acid sequences encoded by the DNAs of the present invention are useful to design new drugs, more specific and therefore with a higher pharmacological potency.
- the novel molecules identified by the screening methods according to the invention are low molecular weight organic molecules, in which case a composition or pharmaceutical composition can be prepared thereof for oral intake, such as in tablets.
- a composition or pharmaceutical composition comprising the nucleic acid molecules, vectors, polypeptides, antibodies and compounds identified by the screening methods described herein, can be prepared for any route of administration including, but not limited to, oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal.
- the nature of the carrier or other ingredients will depend on the specific route of administration and particular embodiment of the invention to be administered. Examples of techniques and protocols that are useful in this context are, inter alia, found in Remington's Pharmaceutical Sciences, 16 th edition, Osol, A (ed.), 1980, which is incorporated herein by reference in its entirety.
- the dosage of these low molecular weight compounds will depend on the disease state or condition to be treated and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound.
- For treating human or animals between approximately 0.5 mg/kg of body weight to 500 mg/kg of body weight of the compound can be administered. Therapy is typically administered at lower dosages and is continued until the desired therapeutic outcome is observed.
- the present compounds and methods including nucleic acid molecules, polypeptides, antibodies, compounds identified by the screening methods described herein, have a variety of pharmaceutical applications and may be used, for example, to treat or prevent unregulated cellular growth, such as cancer cell and tumor growth.
- the present molecules are used in gene therapy.
- gene therapy procedures seee.g. Anderson, Science, 1992, 256, 808-813, which is incorporated herein by reference in its entirety.
- the present invention also encompasses a method of agonizing (stimulating) or antagonizing an ion-x natural binding partner associated activity in a mammal comprising administering to said mammal an agonist or antagonist to one of the above disclosed polypeptides in an amount sufficient to effect said agonism or antagonism.
- One embodiment of the present invention is a method of treating diseases in a mammal with an agonist or antagonist of the protein of the present invention comprises administering the agonist or antagonist to a mammal in an amount sufficient to agonize or antagonize ion-x-associated functions.
- Exemplary diseases and conditions amenable to treatment based on the present invention include, but are not limited to, thyroid disorders (e.g. thyreotoxicosis, myxoedema); renal failure; inflammatory conditions (e.g., Crohn's disease); diseases related to cell differentiation and homeostasis; rheumatoid arthritis; autoimmune disorders; movement disorders; CNS disorders (e.g., pain including neuropathic pain, migraine, and other headaches; stroke; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, anxiety, generalized anxiety disorder, post-traumatic-stress disorder, depression, bipolar disorder, delirium, dementia, severe mental retardation; dyskinesias, such as Huntington's disease or Tourette's Syndrome; attention disorders including ADD and ADHD, and degenerative disorders such as Parkinson's, Alzheimer's; movement disorders, including ataxias, supranuclear palsy, etc.); infections, such as viral infections caused by HIV-1 or HIV-2; metabolic and cardiovascular diseases and disorders (e
- Therapeutically effective doses for the compounds described herein can be estimated initially from cell culture and animal models. For example, a dose can be formulated in animal models to achieve a circulating concentration range that initially takes into account the IC50 as determined in cell culture assays. The animal model data can be used to more accurately determine useful doses in humans.
- Plasma half-life and biodistribution of the drug and metabolites in the plasma, tumors and major organs can also be determined to facilitate the selection of drugs most appropriate to inhibit a disorder. Such measurements can be carried out.
- HPLC analysis can be performed on the plasma of animals treated with the drug and the location of radiolabeled compounds can be determined using detection methods such as X-ray, CAT scan and MRI.
- detection methods such as X-ray, CAT scan and MRI.
- Compounds that show potent inhibitory activity in the screening assays, but have poor pharmacokinetic characteristics can be optimized by altering the chemical structure and retesting. In this regard, compounds displaying good pharmacokinetic characteristics can be used as a model.
- Toxicity studies can also be carried out by measuring the blood cell composition.
- toxicity studies can be carried out in a suitable animal model as follows: 1) the compound is administered to mice (an untreated control mouse should also be used); 2) blood samples are periodically obtained via the tail vein from one mouse in each treatment group; and 3) the samples are analyzed for red and white blood cell counts, blood cell composition and the percent of lymphocytes versus polymo ⁇ honuclear cells. A comparison of results for each dosing regime with the controls indicates if toxicity is present.
- the expected daily dose of a hydrophobic pharmaceutical agent is between 1 to 500 mg/day, preferably 1 to 250 mg/day, and most preferably 1 to 50 mg/day.
- Drugs can be delivered less frequently provided plasma levels of the active moiety are sufficient to maintain therapeutic effectiveness. Plasma levels should reflect thepotency of the drug. Generally, the more potent the compound the lower the plasma levels necessary to achieve efficacy.
- Ion-x mRNA transcripts may found in many tissues, including, but not limited to, brain, kidney, colon, small intestine, stomach, testis, placenta, adrenal gland, peripheral blood leukocytes, bone ma ⁇ ow, retina, ovary, fetal brain, fetal liver, heart, spleen, liver, kidney, lung, muscle, thyroid gland, uterus, prostate, skin, salivary gland, and pancreas. Tissues where specific ion-x mRNA transcripts are expressed are identified in the Examples, below.
- Sequences selected from the group consisting of SEQ ID NO:l to SEQ ID NO: 7, and fragments thereof, will, as detailed above, enable screening the endogenous neurotransmitters/hormones/ligands which activate, agonize, or antagonize ion-x and for compounds with potential utility in treating disorders including, but not limited to, thyroid disorders (e.g.
- thyreotoxicosis myxoedema
- renal failure inflammatory conditions (e.g., Crohn's disease); diseases related to cell differentiation and homeostasis; rheumatoid arthritis; autoimmune disorders; movement disorders; CNS disorders (e.g., pain including neuropathic pain, migraine, and other headaches; stroke; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, anxiety, generalized anxiety disorder, post-traumatic-stress disorder, depression, bipolar disorder, delirium, dementia, severe mental retardation; dyskinesias, such as Huntington's disease or Tourette's Syndrome; attention disorders including ADD and ADHD, and degenerative disorders such as Parkinson's, Alzheimer ⁇ ; movement disorders, including ataxias, supranuclear palsy, etc); infections, such as viral infections caused by HIV-1 or HIV-2; metabolic and cardiovascular diseases and disorders (e.g., type 2 diabetes, obesity, anorexia, hypotension, hypertension, thrombosis, myocardial infarction
- ion-x may be useful in the treatment of respiratory ailments such as asthma, where T cells are implicated by the disease. Contraction of airway smooth muscle is stimulated by thrombin. Cicala et al (1999) Br J Pharmacol 126:478-484. Additionally, in bronchiolitis obliterans, it has been noted that activation of thrombin receptors may be deleterious. Hauck et ⁇ /.(1999) Am J Physiol 277:L22-L29. Furthermore, mast cells have also been shown to have thrombin receptors. Cirino et al (1996) J Exp ⁇ vfed 183:821-827.
- Ion-x may also be useful in remodeling of airway structures in chronic pulmonary inflammation via stimulation of fibroblast procollagen synthesis. See, e.g., Chambers et al. (1998) Biochem J 333:121-127; Trejo et al. (1996) J Biol Chem 271 :21536-21541.
- ion-x may be useful in the treatment of unstable angina due to the role of T cells and inflammation. SeeAukrust et al. (1999) Circulation 100:614-620.
- a further example is the treatment of inflammatory diseases, such as psoriasis, inflammatory bowel disease, multiple sclerosis, rheumatoid arthritis, and thyroiditis. Due to the tissue expression profile of ion-x, inhibition of thrombin receptors may be beneficial for these diseases. See, e.g., Morris et al (1996) Ann Rheum Dis 55:841-843.
- NK cells and monocytes are also critical cell types which contribute to the pathogenesis of these diseases. See, e.g., Naldini & Carney (1996) Cell Immunol 172:3542; Hoffman & Cooper (1995) Blood Cells Mol Dis 21:156-167; Colottaet al. (1994) Am J Pathol 144:975- 985.
- ion-x in spleen may suggest that it may play a role in the proliferation of hematopoietic progenitor cells. See DiCuccio et al. (1996) Exp Hematol 24:914-918.
- ion-x may be useful in the treatment of acute and/or traumatic brain injury.
- Astrocytes have been demonstrated to express thrombin receptors Activation of thrombin receptors may be involved in astrogliosis following brain injury. Therefore, inhibition of receptor activity may be beneficial for limiting neuroinflammation.
- Scar formation mediated by astrocytes may also be limited by inhibiting thrombin receptors. See, e.g, Pindon et al. (1998) Eur J Biochem 255:766-774; Ubl & Reiser. (1997) Glia 21:361-369; Grabham & Cunningham (1995) J Neurochem 64:583-591.
- Ion-x receptor activation may mediate neuronal and astrocyte apoptosis and prevention of neurite outgrowth. Inhibition would be beneficial in both chronic and acute brain injury. See, e.g., Donovan et al. (1997) J Neurosci 17:5316-5326; Turgeon et al (1998) J Neurosci 18:6882-6891; Smith-Swintosky et al. (1997) J Neurochem 69:1890-1896; Gill et al. (1998) Brain Res 797:321-327; Suidan et al. (1996) Semin Thromb Hemost 22:125-133.
- ion-8 nucleic acid sequence SEQ ID NO:l, amino acid sequence SEQ ID NO: 8
- ion-9 nucleic acid sequence SEQ ID NO:2, amino acid sequence SEQ ID NO:9
- ion-12 nucleic acid sequence SEQ ID NO: 5, amino acid sequence SEQ ID NO: 12
- ion-14 nucleic acid sequence SEQ ID NO:7, amino acid sequence SEQ ID NO: 14
- Such neurological diseases and disorders include, but are not limited to, schizophrenia, affective disorders, ADHD/ADD (i.e., Attention Deficit- Hyperactivity Disorder/Attention Deficit Disorder), and neural disorders such as Alzheimer's disease, Parkinson's disease, migraine, and senile dementia as well as depression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses, and the like.
- the invention provides genetic screening procedures that entail analyzing a person's genome — in particular their alleles for ion channels of the invention — to determine whether the individual possesses a genetic characteristic found in other individuals that are considered to be afflicted with, or at risk for, developing a mental disorder or disease of the brain that is suspected of having a hereditary component.
- the invention provides a method for determining a potential for developing a disorder affecting the brain in a human subject comprising the steps of analyzing the coding sequence of one or more ion channel genes from the human subject; and determining development potential for the disorder in said human subject from the analyzing step.
- the invention provides a method of screening a human subject to diagnose a disorder affecting the brain or genetic predisposition therefor, comprising the steps of: (a) assaying nucleic acid of a human subject to determine a presence or an absence of a mutation altering the amino acid sequence, expression, or biological activity ofat least one ion channel that is expressed in the brain, wherein the ion channel comprises an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 12, and SEQ ID NO: 14, or an allelic variant thereof, and wherein the nucleic acid co ⁇ esponds to the gene encoding the ion channel; and (b) diagnosing the disorder or predisposition from the presence or absence of said mutation, wherein the presence of a mutation altering the amino acid sequence, expression, or biological activity of allele in the • nucleic acid co ⁇ elates with an increased risk of developing the disorder.
- the ion channel is ion-8, ion-9, ion-12, or ion-14 comprising amino acid sequences set forth in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 12, and SEQ ID NO: 14, or an allelic variant thereof.
- human subject is meant any human being, human embryo, or human fetus. It will be apparent that methods of the present invention will be of particular interest to individuals that have themselves been diagnosed with a disorder affecting the brain or have relatives that have been diagnosed with a disorder affecting the brain.
- screening for an increased risk determination of whether a genetic variation exists in the human subject that co ⁇ elates with a greater likelihood of developing a disorder affecting the brain than exists for the human population as a whole, or for a relevant racial or ethnic human sub-population to which the individual belongs. Both positive and negative determinations (i.e., determinations that a genetic predisposition marker is present or is absent) are intended to fall within the scope of screening methods of the invention.
- the presence of a mutation altering the sequence or expression of at least one ion-8, ion-9, ion-12, or ion-14 ion channel allele in the nucleic acid is co ⁇ elated with an increased risk of developing the disorder, whereas the absence of such a mutation is reported as a negative determination.
- the "assaying" step of the invention may involve any techniques available for analyzing nucleic acid to determine its characteristics, including but not limited to well- known techniques such as single-strand conformation polymo ⁇ hism analysis (SSCP) [Orita et al, Proc Natl. Acad. Sci. USA, 86: 2766-2770 (1989)]; heteroduplex analysis [Whiteet al, Genomics, 12: 301-306 (1992)]; denaturing gradient gel electrophoresis analysis [Fischer et al, Proc. Natl. Acad. Sci.
- SSCP single-strand conformation polymo ⁇ hism analysis
- the assaying step comprises at least one procedure selected from the group consisting of: (a) determining a nucleotide sequence ofat least one codon ofat least one ion-8, ion-9, ion-12, or ion-14 allele of the human subject; (b) performing a hybridization assay to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences; (c) performing a polynucleotide migration assay to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences; and (d) performing a restriction endonuclease digestion to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences.
- the assaying involves sequencing of nucleic acid to determine nucleotide sequence thereof, using any available sequencing technique.
- any available sequencing technique See, e.g., Sanger et al, Proc. Natl. Acad. Sci. (USA), 74: 5463-5467 (1977) (dideoxy chain termination method); Mirzabekov, TIBTECH, 12: 27-32 (1994) (sequencing by hybridization); Drmanac et al, Nature Biotechnology, 16: 54-58 (1998); U.S. Patent No.
- the analysis may entail sequencing of the entire ion-x gene genomic DNA sequence, or portions thereof; or sequencing of the entire receptor coding sequence or portions thereof.
- the analysis may involve a determination of whether an individual possesses a particular allelic variant, in which case sequencing of only a small portion of nucleic acid— enough to determine the sequence of a particular codon characterizing the allelic variant ⁇ is sufficient.
- This approach is appropriate, for example, when assaying to determine whether one family member inherited the same allelic variant that has been previously characterized for another family member, or, more generally, whether a person's genome contains an allelic variant that has been previously characterized and co ⁇ elated with a mental disorder having a heritable component.
- the assaying step comprises performing a hybridization assay to determine whether nucleic acid from the human subject has a nucleotide sequence identical to or different from one or more reference sequences.
- the hybridization involves a determination of whether nucleic acid derived from the human subject will hybridize with one or more oligonucleotides, wherein the oligonucleotides have nucleotide sequences that co ⁇ espond identically to a portion of the ion-x gene sequence taught herein, or that co ⁇ espond identically except for one mismatch.
- the hybridization conditions are selected to differentiate between perfect sequence complementarity and imperfect matches differing by one or more bases.
- nucleic acid derived from the human subject is subjected to gel electrophoresis, usually adjacent to (or co-loaded with) one or more reference nucleic acids, such as reference ion channel- encoding sequences having a coding sequence identical to all or a portion of a sequence selected from the group consisting of SEQ ID NO:l to SEQ ID NO: 7 (or identical except for one known polymo ⁇ hism).
- reference nucleic acids such as reference ion channel- encoding sequences having a coding sequence identical to all or a portion of a sequence selected from the group consisting of SEQ ID NO:l to SEQ ID NO: 7 (or identical except for one known polymo ⁇ hism).
- nucleic acid from the human subject and the reference sequence(s) are subjected to similar chemical or enzymatic treatments and then electrophoresed under conditions whereby the polynucleotides will show a differential migration pattern, unless they contain identical sequences.
- nucleic acid of a human subject is intended to include nucleic acid obtained directly from the human subject (e.g., DNA or RNA obtained from a biological sample such as a blood, tissue, or other cell or fluid sample); and also nucleic acid derived from nucleic acid obtained directly from the human subject.
- nucleic acid obtained directly from the human subject e.g., DNA or RNA obtained from a biological sample such as a blood, tissue, or other cell or fluid sample
- nucleic acid derived from nucleic acid obtained directly from the human subject e.g., DNA or RNA obtained from a biological sample such as a blood, tissue, or other cell or fluid sample
- mutation includes addition, deletion, and/or substitution of one or more nucleotides in the ion-x gene sequence (e.g., as compared to the ion channel-encoding sequences set forth of SEQ ID NOS: 1-7) and other polymo ⁇ hisms that occur in introns (where introns exist) and that are identifiable via sequencing, restriction fragment length polymo ⁇ hism, or other techniques.
- the various activity examples provided herein permit determination of whether a mutation modulates activity of the relevant receptor in the presence or absence of various test substances.
- the invention provides methods of screening a person's genotype with respect to ion channels of the invention, and co ⁇ elating such genotypes with diagnoses for disease or with predisposition for disease (for genetic counseling).
- the invention provides a method of screening for an ion-8, ion-9, ion-12, or ion-14 mental disorder genotype in a human patient, comprising the steps of: (a) providing a biological sample comprising nucleic acid from the patient, the nucleic acid including sequences co ⁇ esponding to said patient's ion-8, ion-9, ion-12, or ion-14 alleles; (b) analyzing the nucleic acid for the presence of a mutation or mutations; (c) determining an ion-8, ion-9, ion-12, or ion-14 genotype from the analyzing step; and (d) co ⁇ elating the presence of a mutation in an ion-8, ion-9, ion-12,
- the biological sample is a cell sample containing human cells that contain genomic DNA of the human subject.
- the analyzing can be performed analogously to the assaying described in preceding paragraphs.
- the analyzing comprises sequencing a portion of the nucleic acid (e.g., DNA or RNA), the portion comprising at least one codon of the ion-8, ion-9, ion-12, or ion-14 alleles.
- the invention also may be practiced by assaying protein of a human subject to determine the presence or absence of an amino acid sequence variation in ion channel protein from the human subject.
- protein analyses may be performed, e.g., by fragmenting ion channel protein via chemical or enzymatic methods and sequencing the resultant peptides; or by Western analyses using an antibody having specificity for a particular allelic variant of the ion channel.
- the invention also provides materials that are useful for performing methods of the invention.
- the present invention provides oligonucleotides useful as probes in the many analyzing techniques described above.
- such oligonucleotide probes comprise 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides that have a sequence that is identical, or exactly complementary, to a portion of a human ion channel gene sequence taught herein (or allelic variant thereof), or that is identical or exactly complementary except for one nucleotide substitution.
- the oligonucleotides have a sequence that co ⁇ esponds in the foregoing manner to a human ion channel coding sequence taught herein, and in particular, the coding sequences set forth in SEQ ID NOS: 1, 2, 5, or 7.
- an oligonucleotide probe of the invention is purified and isolated.
- the oligonucleotide probe is labeled, e.g., with a radioisotope, chromophore, or fluorophore.
- the probe is covalently attached to a solid support. [See generally Ausubel et al. and Sambrook et al, supra.]
- kits comprising reagents that are useful for practicing methods of the invention.
- the invention provides a kit for screening a human subject to diagnose a mental disorder or a genetic predisposition therefor, comprising, in association: (a) an oligonucleotide useful as a probe for identifying polymo ⁇ hisms in a human ion-8, ion-9, ion-12, or ion-14 ion channel gene, the oligonucleotide comprising 6-50 nucleotides that have a sequence that is identical or exactly complementary to a portion of a human ion-8, ion-9, ion-12, or ion-14 gene sequence or ion- 8, ion-9, ion-12, or ion-14 coding sequence, except for one sequence difference selected from the group consisting of a nucleotide addition, a nucleotide deletion, or nucleotide substitution; and (b) a media packaged with the oligonucleotide useful as
- Exemplary information-containing media include printed paper package inserts or packaging labels; and magnetic and optical storage media that are readable by computers or machines used by practitioners who perform genetic screening and counseling services. The practitioner uses the information provided in the media to co ⁇ elate the results of the analysis with the oligonucleotide with a diagnosis. In a prefe ⁇ ed variation, the oligonucleotide is labeled.
- the invention provides methods of identifying those allelic variants of ion channels of the invention that co ⁇ elate with mental disorders.
- the invention provides a method of identifying an ion channel allelic variant that co ⁇ elates with a mental disorder, comprising steps of: (a) providing a biological sample comprising nucleic acid from a human patient diagnosed with a mental disorder, or from the patient's genetic progenitors or progeny; (b) analyzing the nucleic acid for the presence of a mutation or mutations in at least ion channel that is expressed in the brain, wherein the ion channel comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 2, 5, or 7, or an allelic variant thereof, and wherein the nucleic acid includes sequence co ⁇ esponding to the gene or genes encoding the ion channel; (c) determining a genotype for the patient for the ion channel from said analyzing step; and (d) identifying an allelic variant that co ⁇ elate
- chromosomal localization data facilitates identifying an involved ion channel with a chromosomal marker.
- the foregoing method can be performed to co ⁇ elate ion channels of the invention to a number of disorders having hereditary components that are causative or that predispose persons to the disorder.
- the ion channel comprises ion-9 having an amino acid sequence set forth in SEQ ID NO: 9 or an allelic variant thereof.
- polynucleotides that comprise the allelic variant sequences identified by such methods, and polypeptides encoded by the allelic variant sequences, and oligonucleotide and oligopeptide fragments thereof that embody the mutations that have been identified.
- the invention provides a purified and isolated polynucleotide comprising a nucleotide sequence encoding an ion channel allelic variant identified according to the methods described above; and an oligonucleotide that comprises the sequences that differentiate the ion-8, ion-9, ion-12, or ion-14 allelic variant from the sequences set forth in SEQ ID NOS: 1, 2, 5, or 7.
- the invention also provides a vector comprising the polynucleotide (preferably an expression vector); and a host cell transformed or transfected with the polynucleotide or vector.
- the invention also provides an isolated cell line that is expressing the allele variant ion channel polypeptide; purified cell membranes from such cells; purified polypeptide; and synthetic peptides that embody the allelic variation amino acid sequence.
- the invention provides a purified polynucleotide comprising a nucleotide sequence encoding a ion-9 protein of a human that is affected with a mental disorder; wherein said polynucleotide hybridizes to the complement of SEQ ID NO:2 under the following hybridization conditions: (a) hybridization for 16 hours at 42° C in a hybridization solution comprising 50%> formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate and (b) washing 2 times for 30 minutes at 60°C in a wash solution comprising O.lx SSC and 1% SDS; and wherein the polynucleotide encodes a ion-9 amino acid sequence that differs from SEQ ID NO: 9 by at least one residue.
- An exemplary assay for using the allelic variants is a method for identifying a modulator of ion-x biological activity, comprising the steps of: (a) contacting a cell expressing the allelic variant in the presence and in the absence of a putative modulator compound; (b) measuring ion-x biological activity in the cell; and (c) identifying a putative modulator compound in view of decreased or increased ion-x biological activity in the presence versus absence of the putative modulator.
- Table 5 contains the sequences of the polynucleotides and polypeptides of the invention, in addition to exemplary primers useful for cloning said sequences. "X" indicates an unknown amino acid or a gap (absence of amino acid(s)).
- the following amino acid sequence ⁇ SEQ ID NOS. 8> is a predicted amino acid sequence derived from the DNA sequence of SEQ ID NO. 1:
- SEQ ID NOS: 15 and 16> are, respectively, forward and reverse primers for SEQ ID NO: 1.
- ion8.for ⁇ SEQ ID NO: 15> GACCTGTTGTTCAGGGAGCTTT ion ⁇ .rev ⁇ SEQ ID NO: 16> GGACGAAGAGAAAATACCTGTGA
- the following amino acid sequence ⁇ SEQ ID NO. 9> is a predicted amino acid sequence derived from the DNA sequence of SEQ ID NO. 2:
- sequences ⁇ SEQ ID NOS: 17 and 18> are, respectively, forward and reverse primers for SEQ ID NO: 2.
- ion9.for ⁇ SEQ ID NO: 17> ATGTGGCTAACTCCTAACTCTT ion9.rev ⁇ SEQ ID NO:18> GTACTTGAAGAGTTTTGAGGTGT
- the following amino acid sequence ⁇ SEQ ID NO. 10> is a predicted amino acid sequence derived from the DNA sequence of SEQ ID NO. 3:
- SEQ ID NOS: 19 and 20> are, respectively, forward and reverse primers for SEQ ID NO: 3.
- the following amino acid sequence ⁇ SEQ ID NO. 11> is a predicted amino acid sequence derived from the DNA sequence of SEQ ID NO. 4: ⁇ SEQ ID NO : 11>
- sequences ⁇ SEQ ID NOS: 21 and 22> are, respectively, forward and reverse primers for SEQ ID NO: 4.
- ionll.for ⁇ SEQ ID NO: 21> CTCAGGCATTACTGGAAAGACGA ionll.rev ⁇ SEQ ID NO: 22> GCGCAGCATGATATTCTCCATA
- the following amino acid sequence ⁇ SEQ ID NO. 12> is a predicted amino acid sequence derived from the DNA sequence of SEQ ID NO. 5:
- SEQ ID NOS: 23 and 24> are, respectively, forward and reverse primers for SEQ ID NO: 5.
- ionl2.for ⁇ SEQ ID NO: 23> GGTAAATACTACATAGCCACGAT ionl2.rev ⁇ SEQ ID NO: 24> GGCTGCTTTCAGGTTAGACTCT
- the following amino acid sequence ⁇ SEQ ID NO. 13> is a predicted amino acid sequence derived from the DNA sequence of SEQ ID NO. 6: ⁇ SEQ ID NO: 13>
- sequences ⁇ SEQ ID NOS: 25 and 26> are, respectively, forward and reverse primers for SEQ ID NO: 6.
- ionl3.for ⁇ SEQ ID NO: 25> CACATGTTAAAGAAAGATGGCAGA ionl3.rev ⁇ SEQ ID NO: 26> AACATGTACCCATTCTATCAGCAT
- the following amino acid sequence ⁇ SEQ ID NO . 14> is a predicted amino acid sequence derived from the DNA sequence of SEQ ID NO . 7 :
- sequences ⁇ SEQ ID NOS : 27 and 28> are , respectively, forward and reverse primers for SEQ ID NO : 7 . ionl 4 . for ⁇ SEQ ID NO : 27> TTTAAAGAGCCTTAGGTCCTGGA ionl 4 . rev ⁇ SEQ ID NO : 28> GTAGACACTGTGATCCAATAGAA
- the BLAST algorithm Basic Local Alignment Search Tool, is suitable for determining sequence similarity (Altschul et ⁇ l, J. Mol. Biol, 1990, 215, 403-410, which is inco ⁇ orated herein by reference in its entirety).
- Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
- This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length "W" in the query sequence that either match or satisfy some positive valued threshold score "T" when aligned with a word of the same length in a database sequence.
- HSPs high scoring sequence pair
- T is refe ⁇ ed to as the neighborhood word score threshold (Altschul et ⁇ l, supr ). These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension for the word hits in each direction are halted when: 1) the cumulative alignment score falls off by the quantity X from its maximum achieved value; 2) the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or 3) the end of either sequence is reached.
- the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
- the BLAST algorithm Kerlin et al, Proc. Natl. Acad. Sci. USA, 1993, 90, 5873- 5787, which is inco ⁇ orated herein by reference in its entirety
- Gapped BLAST Altschul et al, Nuc. Acids Res., 1997, 25, 3389-3402, which is inco ⁇ orated herein by reference in its entirety
- P(N) the smallest sum probability
- a nucleic acid is considered similar to an ion channel gene or cDNA if the smallest sum probability in comparison of the test nucleic acid to an ion channel nucleic acid is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
- the Celera database was searched with the NCBI program BLAST (Altschul et ⁇ /, Nuc. Acids Res., 1997, 25, 3389, which is inco ⁇ orated herein by reference in its entirety), using the known protein sequences of ion channels from the SWISSPROT database as query sequences to find patterns suggestive of novel ion channels.
- BLAST programs TBLASTN was used to compare protein sequences to the DNA database dynamically translated in six reading frames.
- a second search strategy was developed using a hidden Markov model (HMM)(Krogh, A., Brown, B., Mian, IS., Sjolander, K and D.Haussler, Hidden Markov models in computational biology: applications to protein modeling.
- HMM hidden Markov model
- HMMs describe the probability distributionof conserved sequence when compared to a related protein family. Because of this different search algorithm, the use of HMMs may yield different and possibly more relevant results than are generated by the BLAST search. Positive hits were further analyzed with the program BLASTX against the non-redundant protein and nucleotide databases maintained at NCBI to determine which hits were most likely to encode novel ion channels, using the standard (default) parameters. This search strategy, together with the insight of the inventors, identified SEQ NO:l to SEQ ID NO: 7 as candidate sequences.
- DNA fragments co ⁇ esponding to a portion of SEQ ID NO:l to SEQ ID NO:7, or complementary nucleotide sequence thereof can be used as probes for hybridization screening of a phage, phagemid, or plasmid cDNA library.
- the DNA fragments are amplified by PCR.
- the PCR reaction mixture of 50 ⁇ l contains polymerase mixture (0.2 mM dNTPs, lx PCR Buffer and 0.75 ⁇ l Expand High Fidelity Polymerase (Roche Biochemicals)), lOOng to l ⁇ g of human cDNA, and 50 pmoles of forward primer and 50 pmoles of reverse primer.
- Primers may be readily designed by those of skill in the art based on the nucleotide sequences provided herein. Amplification is performed in an Applied Biosystems PE2400 thermocycler using for example, the following program: 95°C for 15 seconds, 52°C for 30 seconds and 72° for 90 seconds; repeated for 25 cycles. The actual PCR conditions will depend, for example on the physical characteristics of the oligonucleotide primers and the length of the PCR product. The amplified product can be separated from the plasmid by agarose gel electrophoresis, and purified by QiaquickTM gel extraction kit (Qiagen).
- a lambda phage library containing cDNAs cloned into lambda ZAPII phage- vector is plated with E. coli XL-1 blue host, on 15 cm LB-agar plates at a density of 50,000 pfu per plate, and grown overnight at 37°C; (plated as described by Sambrook et al, supra).
- Phage plaques are transfe ⁇ ed to nylon membranes (Amersham Hybond NJ), denatured for 2 minutes in denaturation solution (0.5 M NaOH, 1.5 M NaCl), renatured for 5 minutes in renaturation solution (1 M Tris pH 7.5, 1.5 M NaCl), and washed briefly in 2xSSC (20x SSC: 3 M NaCl, 0.3 M Na-citrate). Filter membranes are dried and incubated at 80°C for 120 minutes to cross-link the phage DNA to the membranes. The membranes are hybridized with a DNA probe prepared as described above.
- a DNA fragment (25 ng) is labeled with ⁇ - 32 P-dCTP (HEN) using RediprimeTM random priming (Amersham Pharmacia Biotech), according to manufacturers instructions.
- Labeled DNA is separated from uninco ⁇ orated nucleotides by S200 spin columns (Amersham Pharmacia Biotech), denatured at 95°C for 5 minutes and kept on ice.
- the DNA-containing membranes (above) are pre-hybridized in 50 ml ExpressHybTM (Clontech) solution at 68°C for 90 minutes. Subsequently, the labeled DNA probe is added to the hybridization solution, and the probe is left to hybridize to the membranes at 68°C for 70 minutes.
- the membranes are washed five times in 2x SSC, 0.1%> SDS at 42°C for 5 minutes each, and finally washed 30 minutes in O.lx SSC, 0.2% SDS.
- Filters are exposed to Kodak XAR film (Eastman Kodak Company, Rochester, NY., USA) with an intensifying screen at-80°C for 16 hours.
- Kodak XAR film Eastman Kodak Company, Rochester, NY., USA
- One positive colony is isolated from the plates, and replated with about 1000 pfu on a 15 cm LB plate. Plating, plaque lift to filters, and hybridization are performed as described above. About four positive phage plaques may be isolated form this secondary screening.
- cDNA containing plasmids (pBluescript SK-) are rescued from the isolated phages by in vivo excision by culturing XL-1 blue cells co-infected with the isolated phages and with the Excision helper phage, as described by the manufacturer (Stratagene).
- XL-blue cells containing the plasmids are plated on LB plates and grown at 37°C for 16 hours. Colonies (18) from each plate are re-plated on LB plates and grown.
- One colony from each plate is stricken onto a nylon filter in an ordered a ⁇ ay, and the filter is placed on a LB plate to raise the colonies.
- the filter is hybridized with a labeled probe as described above.
- Plasmid DNA is isolated from the three clones by Qiagen Midi Kit (Qiagen) according to the manufacturer's instructions.
- the size of the insert is detennined by digesting the plasmid with the restriction enzymes Notl and Sail, which establishes an insert size.
- the clones are sequenced directly using an ABI377 fluorescence-based sequencer (Perkin-Elmer/ Applied Biosystems Division, PE/ABD, Foster City, CA) and the ABI PRISMTM Ready Dye-Deoxy Terminator kit with Taq FSTM polymerase.
- Each ABI cycle sequencing reaction contains about 0.5 ⁇ g of plasmid DNA. Cycle-sequencing is performed using an initial denaturation at 98°C for 1 minute, followed by 50 cycles using the following parameters: 98°C for 30 seconds, annealing at 50°C for 30 seconds, and extension at 60X2 for 4 minutes. Temperature cycles and times are controlled by a Perkin-Elmer 9600 thermocycler.
- Extension products are purified using CentriflexTMgel filtration cartridges (Advanced Genetic Technologies Co ⁇ ., Gaithersburg, MD). Each reaction product is loaded by pipette onto the column, which is centrifuged in a swinging bucket centrifuge (Sorvall model RT6000B tabletop centrifuge) at 1500 xg for 4 minutes at room temperature. Column-purified samples are dried under vacuum for about 40 minutes and dissolved in 5 ⁇ l of DNA loading solution (83% deionized formamide, 8.3 mM EDTA, and 1.6 mg/ml Blue Dextran). The samples are heated to 90°C for three minutes and loaded into the gel sample wells for sequence analysis using the ABI377 sequencer.
- Sequence analysis is performed by importing ABI377 files into the Sequencer program (Gene Codes, Ann Arbor, MI). Generally, sequence reads of up to about 700 bp are obtained. Potential sequencing e ⁇ ors are minimized by obtaining sequence information from both DNA strands and by re-sequencing difficult areas using primers annealing at different locations until all sequencing ambiguities are removed.
- Ion channel expression patterns can be determined through northern blot analysis of mRNA from different cell and tissue types. Typically, "blots" of isolated mRNA from such cells or tissues are prepared by standard methods or purchased, from commercial suppliers, and are subsequently probed with nucleotide probes representing a fragment of the polynucleotide encoding the ion channel polypeptide.
- probes are labeled radioactively with the use of 32 P-dCTP by RediprimeTM DNA labeling system (Amersham Pharmacia) so as to permit detection during analysis.
- the probe is further purified on a Nick Column (Amersham Pharmacia).
- a multiple human tissue northern blot from Clontech (Human II # 7767-1) is used in hybridization reactions with the probe to determine which tissues express ion channels.
- Pre- hybridization is carried out at 42°C for 4 hours in 5x SSC, lx Denhardt's reagent, 0.1% SDS, 50% formamide, 250 ⁇ g/ml salmon sperm DNA.
- Hybridization is performed overnight at 42°C in the same mixture with the addition of about 1.5xl0 6 cpm/ml of labeled probe.
- the filters are washed several times at 42°C in 0.2x SSC, 0.1% SDS.
- Example 5 Expression of Ion Channel Polypeptides in Mammalian Cells 1. Expression of ion channel polypeptides in 293 cells
- a plasmid bearing the relevant ion channel coding sequence is prepared, using vector pCDNA6 (Invitrogen).
- Vector pCDNA6 contains the CMV promoter and a blasticidin resistant gene for selection of stable transfectants.
- Many other vectors can be used containing, for example, different promoters, epitope tags for detection and/or purification of the protein, and resistance genes.
- the forward primer for amplification of this ion channel polypeptide encoding cDNA is determined by procedures as well known in the art and preferably contains a 5' extension of nucleotides to introduce a restriction cloning site not present in the ion channel cDNA sequence, for example, a Hindlll restriction site and nucleotides matching the ion channel nucleotide sequence.
- the reverse primer is also determined by procedures known in the art and preferably contains a 5' extension of nucleotides to introduce a restriction cloning site not present in the ion channel cDNA sequence, for example, an Xhol restriction site, and nucleotides co ⁇ esponding to the reverse complement of the ion channel nucleotide sequence.
- the PCR conditions are determined by the physical properties of the oligonucleotide primer and the length of the ion channel gene.
- the PCR product is gel purified and cloned into the Hindlll-Xhol sites of the vector
- the plasmid DNA is purified using a Qiagen plasmid mini-prep kit and transfected into, for example, 293 cells using DOTAP transfection media (Boehringer Mannhein, Indianapolis, IN). Transiently transfected cells are tested for ion channel activity and expression after 24-48 hours by established techniques of electrophysiology Electrophysiology, A Practical Approach, DI Wallis editor, IRL Press at Oxford University Press, (1993), and Voltage and patch Clamping with Microelectrodes, TG Smith, H Lecar, SJ Redman and PW Gage, eds., Waverly Press, Inc for the American Physiology Society (1985). This provides one means by which ion channel activity can be characterized.
- DNA is purified using Qiagen chromatography columns and transfected into 293 cells using DOTAP transfection media (Boehringer Mannheim, Indianapolis, IN). Transiently transfected cells are tested for expression after 24 hours of transfection, using Western blots probed with anti-His and anti-ion channel peptide antibodies. Permanently transfected cells are selected with Zeocin and propagated. Production of the recombinant protein is detected from both cells and media by western blots probed with anti-His, anti-Myc or anti-ion channel peptide antibodies.
- a polynucleotide molecule having a nucleotide of SEQ ID NO:l to SEQ ID NO: 7, or complementary nucleotide sequences thereof can be cloned into vector p3-CI.
- This vector is a pUCl 8-derived plasmid that contains the HCMV (human cytomegalovirus) intron located upstream from the bGH (bovine growth hormone) polyadenylation sequence and a multiple cloning site.
- the plasmid contains the dhrf (dihydrofolate reductase) gene which provides selection in the presence of the drug methotrexane (MTX) for selection of stable transformants.
- dhrf dihydrofolate reductase
- MTX drug methotrexane
- Many other vectors can be used containing, for example, different promoters, epitope tags for detection and/or purification of the protein, and resistance genes.
- the forward primer is determined by procedures known in the art and preferably contains a 5' extension which introduces an Xbal restriction site for cloning, followed by- nucleotides which co ⁇ espond to a nucleotide sequence given in SEQ ID NO:l to SEQ ID NO: 7, or portion thereof.
- the reverse primer is also determined by methods well known in the art and preferably contains a 5'- extension of nucleotides which introduces aSall cloning site followed by nucleotides which co ⁇ espond to the reverse complement of a nucleotide sequence given in SEQ ID NOS : 1 -7, or portion thereof.
- the PCR consists of an initial denaturation step of 5 min at 95°C, 30 cycles of 30 sec denaturation at 95°C, 30 sec annealing at 58°C and 30 sec extension at 72°C, followed by 5 min extension at 72°C.
- the PCR product is gel purified and ligated into the.XZ> ⁇ / and Sail sites of vector p3-CI.
- This construct is transformed intoE. coli cells for amplification and DNA purification.
- the DNA is purified with Qiagen chromatography columns and transfected into COS 7 cells using LipofectamineTM reagent (Gibco/BRL), following the manufacturer's protocols. Forty-eight and 72 hours after transfection, the media and the cells are tested for recombinant protein expression.
- Ion channel polypeptides expressed in cultured COS cells can be purified by disrupting cells via homogenization and purifying membranes by centrifugation, solubilizing the protein using a suitable detergent, and purifying the protein by, for example, chromatography. Purified ion channel is concentrated to 0.5 mg/ml in an Amicon concentrator fitted with a YM-10 membrane and stored at -80°C.
- Example 6 Expression of Ion Channel Polypeptides in Insect Cells
- a polynucleotide molecule having a sequence selected from the group consisting of SEQ ID NOS: 1-7, or a portion thereof, or complement thereof is amplified by PCR.
- the forward primer is detennined by methods known in the art and preferably constitutes a 5' extension adding a Ndel cloning site, followed by nucleotides which co ⁇ esponding to a nucleotide sequence provided in SEQ ID NOS: 1-7, or a portion thereof.
- the reverse primer is also determined by methods known in the art and preferably constitutes a 5' extension which introduces aKpnl cloning site, followed by nucleotides which co ⁇ espond to the reverse complement of a nucleotide sequence provided in SEQ ID NOS: 1-7, or a portion thereof.
- the PCR product is gel purified, digested with Ndel and Kpnl, and cloned into the co ⁇ esponding sites of vector pACHTL-A (Pharmingen, San Diego, CA).
- the pAcHTL expression vector contains the strong polyhedrin promoter of ' the Autogr ⁇ ph ⁇ c ⁇ lifornic ⁇ nuclear polyhedrosis virus (AcMNPV), and a lOXHis tag upstream from the multiple cloning site.
- a protein kinase site for phosphorylation and a thrombin site for excision of the recombinant protein preceding the multiple cloning site is also present.
- baculovirus vectors can be used in place of pAcHTL-A, such as pAc373, pVL941 and pAcIMl .
- suitable vectors for the expression of ion channel polypeptides can be used, provided that such vector constructs include appropriately located signals for transcription, translation, and trafficking, such as an in-frame AUG and a signal peptide, as required.
- Such vectors are described in Luckow et ⁇ l., Virology, 1989, 170, 31-39, among others.
- the virus is grown and isolated using standard baculovirus expression methods, such as those described in Summers et ⁇ l., A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987).
- pAcHLT-A containing the gene encoding the ion channel polypeptides is introduced into baculovirus using the "BaculoGold " transfection kit (Pharmingen, San Diego, CA) using methods provided by the manufacturer.
- Individual virus isolates are analyzed for protein production by radiolabeling infected cells with 35 S- methionine at 24 hours post infection. Infected cells are harvested at 48 hours post infection, and the labeled proteins are visualized by SDS-PAGE autoradiography. Viruses exhibiting high expression levels can be isolated and used for scaled up expression.
- a polynucleotide molecule having a sequence of SEQ ID NOS : 1 -7, or a portion thereof is amplified by PCR using the primers and methods described above for baculovirus expression.
- the ion channel polypeptide encoding cDNA insert is cloned into vector pAcHLT-A (Pharmingen), between the Ndel and Kpnl sites (after elimination of an internal Ndel site). DNA is purified using Qiagen chromatography columns.
- the interaction trap/two-hybrid library screening method can be used. This assay was first described in Fields, et ⁇ l, Nature, 1989, 340, 245, which is inco ⁇ orated herein by reference in its entirety. A protocol is published in Current Protocols in Molecular Biology 1999, John Wiley & Sons, NY, and Ausubel, F.M. et al. ⁇ 992,Short Protocols in Molecular Biology, 4 th ed., Greene and Wiley-Interscience, NY, both of which are inco ⁇ orated herein by reference in their entirety. Kits are available from Clontech, Palo Alto, CA (Matchmaker Two Hybrid System 3).
- a fusion of the nucleotide sequences encoding all or a partial ion channel polypeptide and the yeast transcription factor GAL4 DNA-binding domain is constructed in an appropriate plasmid (i.e., pGBKT7), using standard subcloning techniques.
- a GAL4 active domain (AD) fusion library is constructed in a second plasmid (i.e., pGADT7) from cDNA of potential ion channel pofypeptide-binding proteins (for protocols on forming cDNA libraries, see Sambrook et al, supra.
- the DNA-BD/ ion channel fusion construct is verified by sequencing, and tested for autonomous reporter gene activation and cell toxicity, both of which would prevent a successful two-hybrid analysis. Similar controls are performed with the AD/library fusion construct to ensure expression in host cells and lack of transcriptional activity.
- Yeast cells are transformed (ca. 10 5 transformants/mg DNA) with both the ion channel and library fusion plasmids according to standard procedure (Ausubel, et al, supra). In vivo binding of DNA-BD/ ion channel with AD/library proteins results in transcription of specific yeast plasmid reporter genes (i.e., lacZ, HIS3, ADE2, LEU2).
- Yeast cells are plated on nutrient-deficient media to screen for expression of reporter genes. Colonies are dually assayed for ⁇ -galactosidase activity upon growth in Xgal (5-bromo-4- chloro-3-indolyl- ⁇ -D-galactoside) supplemented media (filter assay for ⁇ -galactosidase activity is described in Breeden, et al, Cold Spring Harb. Symp. Quant. Biol. ,1985, 50, 643, which is inco ⁇ orated herein by reference in its entirety).
- FRET fluorescence resonance energy transfer
- CFP Cyan Fluorescent Protein
- YFP Yellow Fluorescent Protein
- a fusion of the nucleotide sequences encoding whole or partial ion channel polypeptides and CFP is constructed in an appropriate plasmid, using standard subcloning techniques.
- a nucleotide encoding a YFP fusion of the possibly interacting target protein is constructed in a second plasmid.
- the CFP/ion channel polypeptide fusion construct is verified by sequencing. Similar controls are performed with the YFP/target protein construct.
- the expression of each protein can be monitored using fluorescence techniques (e.g., fluorescence microscopy or fluorescence spectroscopy).
- Host cells are transformed with both the CFP/ ion channel polypeptide and YFP/target protein fusion plasmids according to standard procedure.
- In situ interactions between CFP/ion channel polypeptide and the YFP/target protein are detected by monitoring the YFP fluorescence after exciting the CFP fluorophore.
- the fluorescence is monitored using fluorescence microscopy or fluorescence spectroscopy.
- changes in the interaction due toe.g, external stimuli are measured using time-resolved fluorescence techniques.
- a YFP fusion library may be constructed from cDNA of potential ion channel polypeptide-binding proteins (for protocols on forming cDNA libraries, see Sambrook et al, supra). Host cells are transformed with both the CFP/ion channel polypeptide and YFP fusion library plasmids. Clones exhibiting FRET are then isolated and the protein interacting with an ion channel polypeptide is identified by rescuing and sequencing the DNA encoding the YFP/target fusion protein.
- Example 9 Assays to Identify Modulators of Ion Channel Activity
- modulators agonists and antagonists
- the following assays typically measure calcium flux, it is contemplated that measurement of other ions may be made.
- modulators that can be identified by these assays are natural ligand compounds of the ion channel; synthetic analogs and derivatives of natural ligands; antibodies, antibody fragments, and/or antibody-like compounds derived from natural antibodies or from antibody-like combinatorial libraries; and/or synthetic compounds identified by high-throughput screening of libraries; and the like. All modulators that bind ion channel are useful for identifying such ion .channels in tissue samples (e.g., for diagnostic pu ⁇ oses, pathological pu ⁇ oses, and the like).
- Agonist and antagonist modulators are useful for up-regulating and down-regulating ion channel activity, respectively, to treat disease states characterized by abnormal levels of ion channels.
- the assays may be performed using single putative modulators, and/or may be performed using a known agonist in combination with candidate antagonists (or visa versa).
- cells e.g., CHO cells
- a construct that encodes the photoprotein apoaequorin In the presence of the cofactor coelenterazine, apoaequorin will emit a measurable luminescence that is proportional to the amount of intracellular (cytoplasmic) free calcium.
- cytoplasmic intracellular free calcium
- ion channel nucleic acid is subcloned into the commercial ⁇ expression vector pzeoSV2 (Invitrogen) and transiently co-transfected along with a construct that encodes the photoprotein apoaquorin (Molecular Probes, Eugene, OR) into CHO cells using the transfection reagent FuGENE 6 (Boehringer-Mannheim) and the transfection protocol provided in the product insert.
- the cells are cultured for 24 hours at 37°C in MEM (Gibco/BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 ⁇ g/ml streptomycin, at which time the medium is changed to serum-free MEM containing 5 ⁇ M coelenterazine (Molecular Probes, Eugene, OR). Culturing is then continued for two additional hours at 37°C. Subsequently, cells are detached from the plate using VERSENE (Gibco/BRL), washed, and resuspended at 200,000 cells/ml in serum-free MEM.
- MEM Gibco/BRL, Gaithersburg, MD
- VERSENE Gibco/BRL
- Dilutions of candidate ion channel modulator compounds are prepared in serum-free MEM and dispensed into wells of an opaque 96-well assay plate at 50 ⁇ l/well. Plates are then loaded onto an MLX microtiter plate luminometer (Dynex Technologies, Inc., Chantilly, VA). The instrument is programmed to dispense 50 ⁇ l cell suspensions into each well, one well at a time, and immediately read luminescence for 15 seconds. Dose-response curves for the candidate modulators are constructed using the area under the curve for each light signal peak. Data are analyzed with Slide Write, using the equation for a one-site ligand, and EC 50 values are obtained. Changes in luminescence caused by the compounds are considered indicative of modulatory activity.
- Changes in intracellular calcium levels are another recognized indicator of ion channel activity, and such assays can be employed to screen for modulators of ion channel activity.
- CHO cells stably transfected with an ion channel expression vector are plated at a density of 4 x 10 4 cells/well in Packard black- walled, 96-well plates specially designed to discriminate fluorescence signals emanating from the various wells on the plate.
- D-PBS modified Dulbecco's PBS
- fetal bovine serum containing 36 mg/L pyruvate and 1 g/L glucose
- calcium indicator dyes Fluo-3TM AM, Fluo-4TM AM, Calcium GreenTM- 1 AM, or Oregon GreenTM 488 BAPTA-1 AM
- Plates are washed once with modified D-PBS without 1%> fetal bovine serum and incubated for 10 minutes at 37°C to remove residual dye from the cellular membrane.
- a series of washes with modified D-PBS without 1% fetal bovine serum is performed immediately prior to activation of the calcium response.
- a calcium response is initiated by the addition of one or more candidate receptor agonist compounds, calcium ionophore A23187 (lO ⁇ M; positive control), or ATP (4 ⁇ M; positive control). Fluorescence is measured by Molecular Device's FLIPR with an argon laser (excitation at 488nm). (See, e.g., Kuntzweiler et al, Drug Development Research, 44(1): 14-20 (1998)). The F-stop for the detector camera was set at 2.5 and the length of exposure was 0.4 milliseconds. Basal fluorescence of cells was measured for 20 seconds prior to addition of candidate agonist, ATP, or A23187, and the basal fluorescence level was subtracted from the response signal. The calcium signal is measured for approximately 200 seconds, taking readings every two seconds. Calcium ionophore A23187 and ATP increase the calcium signal 200% above baseline levels.
- CHO cells transfected with an ion channel expression vector are seeded into 12 mm capsule cups (Molecular Devices Co ⁇ .) at 4 x 10 5 cells/cup in MEM supplemented with 10% fetal bovine serum, 2mM L-glutamine, 10 U/ml penicillin, and 10 ⁇ g/ml streptomycin. The cells are incubated in this medium at 37°C in 5% C0 2 for 24 hours.
- Extracellular acidification rates are measured using a Cytosensor microphysiometer (Molecular Devices Co ⁇ .).
- the capsule cups are loaded into the sensor chambers of the microphysiometer and the chambers are perfused with running buffer (bicarbonate-free MEM supplemented with 4mM L-glutamine, 10 units/ml penicillin, 10 ⁇ g/ml streptomycin, 26mM NaCl) at a flow rate of lOO ⁇ l/minute.
- running buffer bicarbonate-free MEM supplemented with 4mM L-glutamine, 10 units/ml penicillin, 10 ⁇ g/ml streptomycin, 26mM NaCl
- Candidate agonists or other agents are diluted into the running buffer and perfused through a second fluid path. During each 60-second pump cycle, the pump is run for 38 seconds and is off for the remaining 22 seconds.
- the pH of the running buffer in the sensor chamber is recorded during the cycle from 43-58 seconds, and the pump is re-started at 60 seconds to start the next cycle.
- the rate of acidification of the running buffer during the recording time is calculated by the Cytosoft program. Changes in the rate of acidification are calculated by subtracting the baseline value (the average of 4 rate measurements immediately before addition of a modulator candidate) from the highest rate measurement obtained after addition of a modulator candidate.
- the selected instrument detects 61mV/pH unit. Modulators that act as agonists of the ion channel result in an increase in the rate of extracellular acidification compared to the rate in the absence of agonist. This response is blocked by modulators which act as antagonists of the ion channel.
- Example 10 High throughput screening for modulators of ion channels using FLIPR
- FLIPR Fluorometric Imaging Plate Reader This system was developed by Dr. Vince Groppi of the Pharmacia Co ⁇ oration to perform cell- based, high-throughput screening (HTS) assays measuring, for example, membrane potential. Changes in plasma membrane potential co ⁇ elate with the modulation of ion channels as ions move into or out of the cell. The FLIPR system measures such changes in membrane potential.
- a cell- membrane permeant fluorescent indicator dye suitable for measuring changes in membrane potential such as diBAC (bis-(l,3-dibutylbarbituric acid) pentamethine oxonol, Molecular Probes).
- diBAC bis-(l,3-dibutylbarbituric acid) pentamethine oxonol, Molecular Probes.
- COS cells that have been transfected with an ion channel gene of interest are bathed in diBAC. Due to the presence of both endogenous potassium channels in the cells as well as the transfected channel, the addition of 30mM extracellular potassium causes membrane depolarization which results in an increase in diBAC uptake by the cell, and thus an overall increase in fluorescence.
- a potassium channel opener such as chromakalim
- the membrane is hype ⁇ olarized, causing a net outflow of diBAC, and thus a reduction in fluorescence. In this manner the effect of unknown test compounds on membrane potential can be assessed using this assay.
- Tissue specific expression of the cDNAs encoding ions 8-14 was detected using a PCR-based system.
- BLAST results containing the protein sequence alignments obtained from searches of the Celera genomic DNA databases were used to estimate where intron/exon boundaries existed.
- Oligonucleotide primer pairs were designed based on this information to amplify 60 to 800 bp fragments of the predicted coding sequences.
- Primers were synthesized by Sigma-Genosys (The Woodlands, Texas), resuspended in water and the concentration determined by absorbence at 260nm and the concentration adjusted to 25 or 50 ⁇ M with lOmM TrisHCl pH 8.0.
- Primer pairs were tested by PCR using genomic DNA as a template in a lOO ⁇ L reaction mixture containing: 0.5 ⁇ M each forward and reverse primer, lx PCR buffer II (Perkin-Elmer), 1.5mM MgCl 2 (Perkin-Elmer), 0.2mM each dNTP (Gibco-BRL), 0.5 ⁇ g human genomic DNA (Clontech) and 5 units AmpliTaq Gold (Perkin-Elmer) with the following thermocycling conditions in a Perkin-Elmer 9600 thermocycler: one cycle of 95°C for 10 minutes; 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 12X2, for 1 minute; one cycle of 72°C for 10 minutes followed by a 4°C soak.
- Primer pairs that passed the design and testing phase were used to amplify predicted exon sequences from cDNAs using PCR from human tissue cDNA panels obtained from OriGene (Rockville, MD). Expression profiling PCR reactions were set-up as described above except that two concentrations of cDNA were used (1.0 or 0.1 ⁇ L of cDNA) in place of genomic DNA. The amplification conditions used were as follows: one cycle of 95°C for 10 minutes, 35 cycles of 94°C for 30 seconds, 55 ⁇ for 30 seconds, 72°C for 1 minute, one cycle of 72°C for 10 minutes and completed with a 4°C soak. PCR products were analyzed on 2% TAE agarose gels containing 0.5 ⁇ g/mL ethidium bromide. Ion-8
- the forward primer used was to detect expression of ion-8 was:
- Ion-8 mRNA was detected in brain, heart, muscle, peripheral blood leukocytes, and retina. This indicates that compounds modulating the activity of ion-8 may be useful in the treatment of diseases including Alzheimer's disease, Parkinson's disease, schizophrenia, depression, anxiety, migraine, epilepsy, obesity, bipolar and other mood disorders, inflammatory bowel disease, dia ⁇ hea or constipation, asthma, arthritis, leukemias and lymphomas, neurodegeneration, or retinal degeneration.
- Ion-9 The forward primer used was to detect expression of ion-9 was:
- This primer set will prime the synthesis of a 96 base pair fragment in the presence of the appropriate cDNA.
- Ion-9 mRNA was detected in brain, fetal brain, heart, liver, spleen, testis, peripheral blood leukocytes, and retina. This pattern of expression indicates that compounds modulating the activity of ion-9 may be useful in the treatment of diseases including Alzheimer's disease, Parkinson's disease, schizophrenia, depression, anxiety, migraine, epilepsy, obesity, bipolar and other mood disorders, neurodegeneration, or retinal degeneration, spermatogenesis, oogenesis, and other fertility disorders.
- Ion-10 The forward primer used was to detect expression of ion-10 was:
- This primer set will prime the synthesis of a 69 base pair fragment in the presence of the appropriate cDNA.
- Ion-10 mRNA was detected in testis, peripheral blood leukocytes, and retina. This indicates that compounds modulating the activity of ion-10 may be useful in the treatment of diseases including inflammatory bowel disease, asthma, arthritis, leukemias and lymphomas, and retinal degeneration.
- Ion-11 The forward primer used was to detect expression of ion- 11 was:
- Ionl 1 mRNA was detected in peripheral blood leukocytes and retina. This pattern of expression indicates that compounds modulating the activity of ion- 11 may be useful in the treatment of diseases including inflammatory bowel disease, asthma, arthritis, leukemias and lymphomas, and retinal degeneration.
- Ion-12 The forward primer used was to detect expression of ion-12 was:
- This primer set will prime the synthesis of a 257 base pair fragment in the presence of the appropriate cDNA.
- Ion-12 mRNA was detected in testis, ovary, peripheral blood leukocytes, bone ma ⁇ ow, fetal brain, and retina. This indicates that compounds modulating the activity of ion- 12 may be useful in the treatment of diseases including Alzheimer's disease, Parkinson's disease, schizophrenia, depression, anxiety, migraine, epilepsy, obesity, bipolar and other mood disorders, inflammatory bowel disease, asthma, arthritis, leukemias and lymphomas, neurodegeneration, or retinal degeneration, spermatogenesis, oogenesis, and other fertility disorders.
- Ion-13 The forward primer used was to detect expression of ion-13 was:
- This primer set will prime the synthesis of a 90 base pair fragment in the presence of the appropriate cDNA.
- Ionl 3 mRNA was detected in peripheral blood leukocytes and retina. This pattern of expression indicates that compounds modulating the activity of ion-13 may be useful in the treatment of diseases including inflammatory bowel disease, asthma, arthritis, leukemias and lymphomas, and retinal degeneration.
- Ion-14 The forward primer used was to detect expression of ion-14 was:
- This primer set will prime the synthesis of a 103 base pair fragment in the presence of the appropriate cDNA.
- Ion-14 mRNA was detected in brain, heart, spleen, liver, kidney, small intestine, lung, muscle, thyroid gland, adrenal gland, ovary, uterus, prostate, skin, fetal brain, fetal liver, stomach, testis, placenta, colon, salivary gland, pancreas, adrenal gland, peripheral blood leukocytes, bone ma ⁇ ow, and retina.
- compounds modulating the activity of ion-14 may be useful in the treatment of diseases including Alzheimer's disease, Parkinson's disease, schizophrenia, depression, anxiety, migraine, epilepsy, obesity, bipolar and other mood disorders, cardiomyopathies, a ⁇ hythmias, hyper- or hypo-thyroidism, deficits in uterine contractility, hype ⁇ rostatism, inflammatory bowel disease, dia ⁇ hea or constipation, diabetes, asthma, arthritis, leukemias and lymphomas, neurodegeneration, or retinal degeneration.
- diseases including Alzheimer's disease, Parkinson's disease, schizophrenia, depression, anxiety, migraine, epilepsy, obesity, bipolar and other mood disorders, cardiomyopathies, a ⁇ hythmias, hyper- or hypo-thyroidism, deficits in uterine contractility, hype ⁇ rostatism, inflammatory bowel disease, dia ⁇ hea or constipation, diabetes, asthma, arthritis, leukemias and lymphomas, neurodegeneration, or retinal degeneration.
- a chimeric receptor can be used to measure the activity of ligand binding when the ligand' s native receptor activity is not amenable to easy measurement.
- Such chimera may consist of a ligand-binding domain of one receptor fused to the pore-forming domain of another receptor.
- a useful example of such a chimera can be found in WO 00/73431 A2.
- the pore-forming transmembrane domain of ion-9 (SEQ ID NO: 2) can be fused, for example, with the extracellular domain of the alpha7 nicotinic acetylcholine receptor to form a chimeric receptor that binds alpha7 receptor ligands but passes cu ⁇ ent like that of ion-9.
- PCR primers are designed to amplify the 5' region of the alpha7 receptor (GenBank accession number U62436) with a region of overlap with ion-9 on the 3'- most primer.
- PCR is performed using the appropriate cDNA clone as a template using Platinum Taq polymerase (Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions.
- the PCR products from these two reactions are then diluted 1 : 1000 and pooled in a second PCR mixture with appropriately designed primers to generate the final chimeric cDNA by splice-overlap PCR.
- These primers also add anEcoRI restriction site to the 5' end and a Notl site to the 3' end to facilitate subcloning into pcDNA3.1 (Invitrogen).
- the PCR product is ligated into pcDNA3.1 and transformed into competentE. coli (Life Technologies, Gaithersburg, MD). Isolated E.
- coli colonies selected on ampicillin-containing medium are isolated and expanded.
- the DNA from the plasmid inE. coli is isolated and sequenced to verify that the expected sequences are obtained.
- the DNA is then transformed into mammalian cells such as SH-EP1 cells using cationic lipid transfection reagent. Cells that are stably transformed are selected in the presence of 800 micrograms/ml geneticin. These cells are then assayed as described supra for changes in intracellular calcium or changes in membrane potential in response to ligands, e.g. nicotine.
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